U.S. Department of Justice

Office of Justice Programs

National Institute of Justice

DNA Short

Tandem

Repeats

Improved Analysis of

With Time-of-Flight

Mass Spectrometry

science and technology research report
U.S. Department of Justice

Office of Justice Programs

810 Seventh Street N.W.

Washington, DC 20531

 John Ashcroft

Attorney General

Office of Justice Programs National Institute of Justice

World Wide Web Site World Wide Web Site 

http://www.ojp.usdoj.gov http://www.ojp.usdoj.gov/nij
IMPROVED ANALYSIS OF DNA SHORT TANDEM REPEATS

WITH TIME-OF-FLIGHT MASS SPECTROMETRY

John M. Butler and Christopher H. Becker

Science and Technology Research Report

October 2001

 NCJ 188292
John M. Butler, Ph.D., is currently a research chemist at the National Institute of Standards and Technology and principle

investigator on an NIJ-funded project to further develop multiplex PCR and time-of-flight mass spectrometry for future forensic

DNA typing assays. He was the first to demonstrate that short tandem repeat typing could be performed with capillary 

electrophoresis.

Christopher H. Becker, Ph.D., is currently senior director of proteomics technology at Thermo Finnigan in San Jose,

California. During the span of this project, he was president and chief operations officer of GeneTrace Systems, Inc.

This project was supported under grant number 97–LB–VX–0003 from the National Institute of Justice, Office of Justice

Programs, U.S. Department of Justice. Points of view in this document are those of the authors and do not necessarily 

represent the official position or policies of the U.S. Department of Justice.

This document is not intended to create, does not create, and may not be relied upon to create any rights, substantive or

procedural, enforceable at law by any party in any matter, civil or criminal.

For further information, contact John M. Butler, National Institute of Standards and Technology, 100 Bureau Drive,

Gaithersburg, MD 20899; phone 301–975–4049; e-mail john.butler@nist.gov.

The National Institute of Justice is a component of the Office of Justice Programs, which also includes the Bureau of

Justice Assistance, the Bureau of Justice Statistics, the Office of Juvenile Justice and Delinquency Prevention, and the

Office for Victims of Crime.

Sarah V. Hart

Director, National Institute of Justice

Lois Tully

Project Monitor
ACKNOWLEDGMENTS

The project described in this

report could not have happened

without the hard work and support of a number of people at GeneTrace

Systems, Inc. First and foremost, Jia Li

did some of the early primer design

and STR work to demonstrate that

STRs could be effectively analyzed by

mass spectrometry. Jia taught us a lot

about PCR and was always encouraging of our work. Likewise, Tom Shaler

was important in the early phases of

this research with his expert advice in

mass spectrometry and data processing.

The first GeneTrace STR mass spectra

were carefully collected by Tom, and

thus he and Jia deserve credit for helping obtain the funding for this study.

Dan Pollart synthesized numerous

cleavable primers for this project, especially in the first year of our work.

David Joo and Wendy Lam also prepared PCR and SNP primers for the

later part of this work. A number of

people assisted in robotic sample preparation and sample cleanup, including

Mike Abbott, Jon Marlowe, David

Wexler, and Rebecca Turincio. Joanna

Hunter, Vera Delgado, and Can Nhan

ran many of the STR samples on the

automated mass spectrometers. Their

hard work made it possible to focus on

experimental design and data analysis

rather than routine sample handling.

It was a great blessing to have talented

and supportive coworkers throughout

the course of this project. Kathy

Stephens, Jia Li, Tom Shaler, Yuping

Tan, Christine Loehrlein, Joanna

Hunter, Hua Lin, Gordy Haupt, and

Nathan Hunt provided useful discussions on a number of issues and helped

develop assay parameters and tackle

automation issues, among other things.

Nathan Hunt was especially important

to the success of this project because

he developed the STR genotyping algorithm and CallSSR software as well as

the multiplex SNP primer design software. Kevin Coopman developed the

SNP genotyping algorithm and calling

software and was always eager to analyze our multiplex SNP samples. Joe

Monforte and Roger Walker served as

our supervisors for the first year and

second year of this project, respectively,

which allowed us the opportunity to

devote sufficient time to doing the work

described in this project. Last but not

least, Debbie Krantz served as an able

administrator of these two NIJ grants

and took care of the financial aspects.

We also were supported with samples

and sequence information from a number of scientific collaborators. Steve Lee

and John Tonkyn from the California

Department of Justice DNA Laboratory

provided genomic DNA samples and

STR allelic ladders. Debang Liu from

Northwestern University provided

the D3S1358 DNA sequence used for

improved primer design purposes.

Peter Oefner and Peter Underhill from

the Department of Genetics at Stanford

University provided male population

samples and Y-chromosome SNP

sequences. The encouragement and

support of Lisa Forman and Richard

Rau from the Office of Justice Programs

at the National Institute of Justice propelled this work from an idea to a

working product. In addition, Dennis

Reeder from the National Institute of

Standards and Technology was always

a constant source of encouragement

at scientific meetings.

iii
CONTENTS

Acknowledgments ........................................................................................................iii

Executive Summary ......................................................................................................1

Introduction ............................................................................................................1

Purpose of the Report ..............................................................................................1

Short Tandem Repeats..............................................................................................2

Single Nucleotide Polymorphisms............................................................................5

Conclusions and Implications ..................................................................................6

Project Description ........................................................................................................9

STR Grant ................................................................................................................9

SNP Grant..............................................................................................................10

Scope and Methodology ..............................................................................................21

Assay Development and Primer Testing..................................................................22

Sample Cleanup and Mass Spectrometry................................................................29

Sample Genotyping ................................................................................................31

Comparison Tests With ABI 310 Genetic Analyzer ................................................33

Results and Discussion of STR Analysis by Mass Spectrometry ....................................35

Marker Selection and Feasibility Studies With STR Loci ........................................35

Multiplex STR Work ..............................................................................................40

Comparison Tests Between ABI 310 and Mass Spectrometry Results......................40

PCR Issues..............................................................................................................41

Analytical Capabilities of This Mass Spectrometry Method ....................................51

Results and Discussion of Multiplex SNPs ..................................................................63

Mitochondrial DNA Work......................................................................................64

Y-Chromosome Work ............................................................................................66

References ....................................................................................................................73

Published Papers and Presentations ............................................................................75

v
vi

List of exhibits included with this report:

Exhibit 1. Schematic of GeneTrace automated time-of-flight mass spectrometer......2

Exhibit 2. PCR product sizes with newly designed primers for commonly used

STR loci compared with commercially available primers used 

in multiplex sets for fluorescence-based assays ....................................3

Exhibit 3. Fluorescent multiplex STR result with AmpF1STR® COfilerTM

kit and ABI 310 Genetic Analyzer ........................................................4

Exhibit 4. Schematic of expected allele masses for a CSF1PO-TPOX-THO1

(CTT) multiplex involving overlapping allele size ranges......................4

Exhibit 5. Mass spectrum of an STR triplex involving TPOX, THO1, 

and CSF1PO ........................................................................................5

Exhibit 6. Mass spectrum of SNP 10-plex assay for screening polymorphic sites 

in the mtDNA control region ................................................................6

Exhibit 7. Overlay of CE electropherograms demonstrating male-specific

amplification with the 17-plex set of PCR primers................................7

Exhibit 8. Mass spectrum of an STR multiplex sample with 

nonoverlapping alleles ..........................................................................9

Exhibit 9. Mass spectrum of STR multiplex mixture of TH01 and TPOX 

allelic ladders ......................................................................................10

Exhibit 10. Mass spectrum of a multiplexed TH01 and TPOX sample showing

interleaved amplicons that are only 85 Da apart ................................10

Exhibit 11. STR data collection times for CDOJ samples ......................................11

Exhibit 12. CDOJ CSF1PO results compared with ABI 310 and mass

spectrometry methods ........................................................................12

Exhibit 13. CDOJ TPOX results compared with ABI 310 and mass

spectrometry methods ........................................................................13

Exhibit 14. CDOJ TH01 STR results compared with ABI 310 and mass

spectrometry methods ........................................................................14

Exhibit 15. CDOJ amelogenin results compared with ABI 310 and mass

spectrometry methods ........................................................................15

Exhibit 16. CDOJ D3S1358 STR results compared with ABI 310 and mass 

spectrometry methods ........................................................................16

Exhibit 17. CDOJ D16S539 STR results compared with ABI 310 and mass

spectrometry methods ........................................................................17

Exhibit 18. CDOJ D7S820 STR results compared with ABI 310 and mass

spectrometry methods ........................................................................18

Exhibit 19. CDOJ FGA STR results compared with ABI 310 and mass 

spectrometry methods ........................................................................19
vii

Exhibit 20. Schematic of the STR assay using the GeneTrace cleavable 

primer technology ..............................................................................21

Exhibit 21. Schematic of the SNP assay using the GeneTrace cleavable 

primer technology ..............................................................................22

Exhibit 22. D3S1358 sequence with PCR primer locations ..................................23

Exhibit 23. Expected mass-to-charge ratios of various ions in the 

mtDNA 10-plex assay ........................................................................24

Exhibit 24. Primer sequences designed for STR markers tested by 

mass spectrometry ..............................................................................24

Exhibit 25. CEPH family pedigrees for samples examined ....................................25

Exhibit 26. Mitochondrial DNA primers used for 10-plex SNP reaction ..............26

Exhibit 27. Multiplex PCR primers used for Y SNP markers ................................27

Exhibit 28. CE electropherograms of “dropout” experiments conducted 

on a 9-plex PCR primer set used in developing Y-chromosome 

SNP markers ......................................................................................28

Exhibit 29. Photo of the automated GeneTrace mass spectrometry ion optics 

over a sample plate containing 384 different DNA samples ................30

Exhibit 30. Mass ladder file for STR loci analyzed in this study ............................31

Exhibit 31. Mass spectra of CDOJ samples amplified with CSF1PO primers ........32

Exhibit 32. Sample sets run on ABI 310 Genetic Analyzer with 

AmpF1STR® ProfilerPlusTM or AmpF1STR® COfilerTM

fluorescent STR kits ............................................................................33

Exhibit 33. Tetranucleotide markers identified through literature and public 

database searches as possible candidates for early STR 

marker development ..........................................................................35

Exhibit 34. STR markers examined at GeneTrace during the course 

of this project as sorted by their chromosomal position......................36

Exhibit 35. Mass spectra for CSF1PO, TPOX, TH01, and amelogenin

using K562 DNA ................................................................................37

Exhibit 36. Mass spectra of STR allelic ladders from CSF1PO, 

TPOX, TH01, and VWA......................................................................38

Exhibit 37. Mass spectra of a D8S1179 sample illustrating the benefit 

of a dideoxynucleotide termination approach ....................................39

Exhibit 38. Multiplexing strategies for STR genotyping using FTATM paper ..........41

Exhibit 39. CDOJ D8S1179 STR results with the mass spectrometry method ......42

Exhibit 40. CDOJ DYS391 STR results with the mass spectrometry method ........43

Exhibit 41. Plot of measured masses versus sample number from 

four different STR loci ........................................................................44
viii

Exhibit 42. Effects of sequence variation on PCR amplification in or 

around STR repeat regions..................................................................45

Exhibit 43. Mass spectra of CDOJ samples amplified with D7S820 primers ........46

Exhibit 44. Electropherograms of ABI 310 results for new STR 

microvariants seen in the Stanford male population samples ..............47

Exhibit 45. ProfilerPlusTM results from Stanford male population samples............48

Exhibit 46. Plot of allele mass offsets (allele 1 versus allele 2) for 

heterozygous samples from four different loci ....................................49

Exhibit 47. Plot of X allele mass offset versus Y allele mass offset for 

88 amelogenin samples ......................................................................49

Exhibit 48. Mass spectra comparing an STR sample amplified with TaqGold

polymerase and Tsp polymerase..........................................................50

Exhibit 49. Mass spectrum demonstrating detection of stutter products from a 

particularly stutter-prone dinucleotide repeat locus ............................50

Exhibit 50. Primer sequence determination with exonuclease digestion 

and mass difference measurements ....................................................51

Exhibit 51. Mass spectrum of AmpF1STR® Green I primer mix ............................51

Exhibit 52. TH01 STR primer positions for commercially available primers 

highlighted on the GenBank sequence ................................................52

Exhibit 53. TPOX STR primer positions for commercially available primers 

highlighted on the GenBank sequence ................................................53

Exhibit 54. CSF1PO STR primer positions for commercially available primers

highlighted on the GenBank sequence ................................................54

Exhibit 55. Mass spectrum of a TH01 allelic ladder reamplified from

AmpF1STR® Green I allelic ladders ....................................................55

Exhibit 56. Mass spectra of D5S818 allelic ladders from two manufacturers ........55

Exhibit 57. Mass spectra of TPOX PCR products from various amounts 

of K562 DNA template material..........................................................56

Exhibit 58. Mass spectra of CDOJ samples amplified with TPOX primers ............57

Exhibit 59. Mass spectra of CDOJ samples amplified with TH01 primers ............58

Exhibit 60. Comparison of allele masses collected 6 months apart........................59

Exhibit 61. Fifteen replicate analyses of a TPOX allelic ladder to measure mass

precision and accuracy........................................................................60

Exhibit 62. Upper strand (TCAT repeat) and lower strand (AATG repeat) 

mass differences for the TH01 allelic ladder........................................60

Exhibit 63. Comparison of ABI 310 and mass spectrometry allele calls for

90 CEPH/diversity samples ................................................................61
ix

Exhibit 64. Histogram of mass difference measurements for 200 samples 

(50 for each ddN) ..............................................................................63

Exhibit 65. Schematic representation of the mtDNA control region 

10-plex SNP assay ..............................................................................64

Exhibit 66. SNP ions impacting multiplex design ................................................64

Exhibit 67. Multiplex PCR information for 17-plex PCR reaction ........................66

Exhibit 68. CE electropherograms showing a multiplex PCR sample

compared with individual PCR reactions ............................................67

Exhibit 69. Primers for testing Y-chromosome SNP markers in singleplexes ........68

Exhibit 70. Y SNP multiplex primer information ..................................................68

Exhibit 71. Human autosomal SNP markers designed for testing NIH 

diversity panel ....................................................................................69

Exhibit 72. Characteristics of STR and SNP markers ............................................70

Exhibit 73. STR genotypes for standard DNA templates K562, AM209, 

and UP006 obtained using AmpF1STR® ProfilerPlusTM and

AmpF1STR® COfilerTM fluorescent STR kits ........................................70

Exhibit 74. An ABI 310 CE electropherogram showing a heterozygous 

sample with a 15.2 microvariant at STR locus D3S1358 ....................71
1

EXECUTIVE SUMMARY

Introduction

The advent of DNA typing and

its use for human identity testing has revolutionized law

enforcement investigations in recent

years by allowing forensic laboratories

to match suspects with minuscule

amounts of biological evidence from a

crime scene. Equally important is the

use of DNA to exclude suspects who

were not involved in a crime or to

identify human remains in an accident. 

The past decade has seen numerous

advances in the DNA testing procedures, most notably among them the

development of PCR (polymerase

chain reaction)-based DNA typing

methods. Technologies for measuring

DNA variations, both length and

sequence polymorphisms, have also

advanced rapidly in the past decade.

The time needed to determine a sample’s DNA profile has dropped from

6–8 weeks to 1–2 days, and with

more recent advancements, the time

needed to process samples may

decrease to as little as a few hours,

maybe even a few minutes.

Simultaneous with the evolution

of DNA markers and technologies

embraced by the forensic community

has been the acceptance and use of

DNA typing information. The courtroom battles over statistical issues

that were common in the late 1980s

and early 1990s have subsided as DNA

evidence has become more widely

accepted. 

In the past 5 years, DNA databases

have emerged as powerful tools for

criminal investigations, much like the

fingerprint databases that have been

used routinely for decades. 

The United Kingdom launched a

nationwide DNA database in 1995

that now contains more than 1 million

DNA profiles of convicted felons—

profiles that have been used to aid

more than 75,000 criminal investigations. National DNA databases are

springing up in countries all over the

world as their value to law enforcement is being recognized. 

In the United States, the FBI has

developed the Combined DNA Index

System (CODIS) with the anticipation

that several million DNA profiles will

be entered into this database in the

next decade. All 50 States now have

laws requiring DNA typing of convicted offenders, typically for violent

crimes such as rape or homicide. 

While the law enforcement community is gearing up to gather millions of

DNA samples from convicted felons,

the DNA typing technology needs

improvement. Large backlogs of samples exist today due to the high cost of

performing the DNA testing and limited capabilities in forensic laboratories.

As of the summer of 1999, several

States, including California, Virginia,

and Florida, had backlogs of more

than 50,000 samples. A need exists for

more rapid and cost-effective methods

for high-throughput DNA analysis to

process samples currently being gathered for large criminal DNA databases

around the world.

At the start of this project in June

1997, commercially available slab gel

or capillary electrophoresis instruments

could handle only a few dozen samples

per day. While larger numbers of samples can be processed by increasing

the number of laboratory personnel

and instruments, the development

of high-throughput DNA processing

technologies promises to be more cost

effective in the long run, especially for

the generation of large DNA databases. GeneTrace Systems, Inc., a small

biotechnology company located in

Alameda, California, has developed

high-throughput DNA analysis capabilities using time-of-flight mass spectrometry coupled with parallel sample

preparation on a robotic workstation.

The GeneTrace technology allows several thousand samples to be processed

daily. DNA samples can be analyzed

in seconds, rather than minutes or

hours, and with improved accuracy

compared with conventional electrophoresis methods. 

Overall, the mass spectrometry method

described in this study is two orders

of magnitude faster in sample processing time than conventional techniques.

Purpose of the Report

This NIJ project was initiated to adapt

the GeneTrace technology to human

identity DNA markers commonly used

by forensic DNA laboratories, specifically short tandem repeat (STR) markers. An extension of the original grant

was submitted in December 1997 to

fund the development of single nucleotide polymorphism (SNP) markers

from mitochondrial DNA and the Y

chromosome. 

Based on the results obtained in this

study, the authors believe mass spectrometry can be a useful and effective

means for high-throughput DNA

analysis, and that it has the capabilities to meet the needs of the forensic

DNA community for offender DNA

databases. 

However, due to limited resources

and a perceived difficulty to enter 

the forensic DNA market, GeneTrace

made a business decision to not pursue this market. While the STR milestones on the original grant were

met, only the initial milestones were

achieved on the SNP portion of the

NIJ grant because of the premature

termination on the part of GeneTrace.

GeneTrace Systems, Inc., developed

an integrated high-throughput DNA

analysis system involving the use of

proprietary chemistry, robotic sample

manipulation, and time-of-flight mass

spectrometry. The purpose of this NIJ

project was to apply the GeneTrace

technology to improve the analysis of

STR markers commonly used in forensic DNA laboratories.
2

EXECUTIVE SUMMARY

Mass spectrometry is a versatile analytical technique that involves the detection of ions and the measurement of

their mass-to-charge ratio. Because

these ions are separated in a vacuum

environment, analysis times can be

extremely rapid, often within microseconds. Many advances have been

made in the past decade for the analysis of biomolecules such as DNA, proteins, and carbohydrates since the

introduction of a new ionization technique known as matrix-assisted laser

desorption-ionization (MALDI) and

the discovery of new matrixes that

effectively ionize DNA without extensive fragmentation. When coupled

with time-of-flight mass spectrometry,

this method for measuring biomolecules is commonly referred to as

MALDI-TOF-MS. A schematic of

MALDI-TOF-MS is presented in

exhibit 1.

Short Tandem Repeats

Short tandem repeat (STR) DNA markers, also referred to as microsatellites or

simple sequence repeats (SSRs), consist

of tandemly repeated DNA sequences

with a core repeat of 2–6 base pairs (bp).

STR markers are readily amplified during PCR by using primers that bind in

conserved regions of the genome flanking the repeat region. Forensic laboratories prefer tetranucleotide loci (i.e.,

4 bp in the repeat) due to the lower

amount of “stutter” produced during

PCR. (Stutter products are additional

peaks that can complicate the interpretation of DNA mixtures by appearing

in front of regular allele peaks.) The

number of repeats can vary from 3 or 4

repeats to more than 50 repeats with

extremely polymorphic markers. The

number of repeats, and hence the size

of the PCR product, may vary among

samples in a population making STR

markers useful in identity testing or

genetic mapping studies.

Shortly after this project was initiated,

the FBI designated 13 core STR loci

for the nationwide CODIS database.

These STR loci are TH01, TPOX,

CSF1PO, VWA, FGA, D3S1358,

D5S818, D7S820, D13S317, D16S539,

D8S1179, D18S51, and D21S11. The

sex-typing marker, amelogenin, is also

included in STR multiplexes that cover

the 13 core STR loci. Each sample

must have these 14 markers tested to

be entered into CODIS.

To illustrate the kinds of numbers

involved to analyze the current national

sample backlog of ~500,000 samples,

more than 7 million genotypes must

be generated. Using currently available technologies, an estimated $25

million (~$50/sample) and more than

5 years for well-trained and well-funded

Acceleration Region

Detector

Ion Extractor

Drift Region

Electric-Field Free

Pulsed Laser Beam

High-Density

Sample Array

DNA Reaction Products

(separated by mass while

drifting to the detector)

X-Y sample

selection

(moves array)

Exhibit 1. Schematic of GeneTrace automated time-of-flight mass 

spectrometer. DNA molecules are liberated from a solid-phase matrix 

environment with a laser pulse. The DNA reaction products are separated by 

size (mass) in a matter of microseconds, as opposed to hours using conventional 

methods. For each run, hundreds of samples are prepared in parallel using a 

robotic workstation and spotted on a sample plate that is introduced to the 

vacuum environment of the mass spectrometer. The sample plate moves under 

the fixed laser beam to allow sequential sample analysis.
3

EXECUTIVE SUMMARY

laboratories would be required to

determine those 7 million genotypes.

With the high cost and effort required,

most of these backlogged samples are

being stored in anticipation of future 

analysis and inclusion in CODIS, pending the development of new, faster

technology or the implementation of

more instruments using the current

electrophoresis technologies. 

Time-of-flight mass spectrometry has

the potential to bring DNA sample

processing to a new level in terms of

high-throughput analysis. However,

there are several challenges to using

MALDI-TOF-MS for the analysis of

PCR products, such as STR markers.

Mass spectrometry resolution and 

sensitivity are diminished when either

the DNA size or the salt content of

the sample is too large. By redesigning

the PCR primers to bind close to the

repeat region, the STR allele sizes

are reduced to benefit the resolution

and sensitivity of the PCR products.

Therefore, much of this project

involved designing and testing new

PCR primers that produced smaller

amplicon sizes for STR markers of

forensic interest. This research focused

on STR loci that have been developed

by commercial manufacturers and studied extensively by forensic scientists.

These include all of the GenePrintTM

tetranucleotide STR systems from

Promega Corporation (Madison, WI)

as well as the 13 CODIS STR loci that

are covered by the Profiler PlusTM and

COfilerTM kits from Applied Biosystems

(ABI) (Foster City, CA) (exhibit 2).

Where possible, primers were designed

to produce amplicons less than 100 bp

in size, although it has been possible

to resolve neighboring STR alleles as

large as 140 bp. For example, TPOX

alleles 6–14 ranged from 69–101 bp in

size with GeneTrace-designed primers;

while with Promega’s GenePrintTM

primers, the same TPOX alleles ranged

in size from 224–256 bp. Unfortunately,

due to the long and complex repeat

structures of several STR markers, this

study was unable to obtain the necessary single-base resolution with the following STR loci: D21S11, D18S51, and

FGA (see Results and Discussion of

STR Analysis by Mass Spectrometry).

To verify the STR results obtained from

the mass spectrometry method, the

authors collaborated with the California

Department of Justice (CDOJ) DNA

Laboratory in Berkeley to generate a

large data set. CDOJ provided 88 samples that had been previously genotyped

STR Locus

Known

Alleles

GeneTrace Sizes

Commerically Available

Sizes*

(newly designed primers

in this study) Applied

Biosystems Promega

Amelogenin X, Y 106, 112 bp 106, 112 bp 212, 218 bp 

CD4 4–15 81–136 bp N.A.

CSF1PO 6–15 87–123 bp 280–316 bp 291–327 bp

F13A1 3–17 112– 168 bp N.A. 279–335 bp

F13B 6–12 110–134 bp N.A. 169–193 bp

FES/FPS 7–15 76–108 bp N.A. 222–254 bp

FGA 15–30 118– 180 bp 206–266 bp N.A.

D3S1358 9–20 76–120 bp 101–145 bp N.A.

D5S818 7–15 89–121 bp 134–166 bp 119–151 bp

D7S820 6–14 66–98 bp 257–289 bp 215–247 bp

D8S1179 8–18 92–130 bp 127–167 bp N.A.

D13S317 7–15 98–130 bp 201–233 bp 165–197 bp

D16S539 5,8–15 81–121 bp 233–273 bp 264–304 bp

D18S51 9–27 120–192 bp 272–344 bp N.A.

D21S11 24–38 150–190 bp 186–242 bp N.A.

DYS19 8–16 76–108 bp N.A.

DYS391 9–12 99–111 bp N.A.

HPRTB 6–17 84–128 bp N.A. 259–303 bp

LPL 7–14 105–133 bp N.A. 105–133 bp

TH01 3–13.3 55–98 bp 160–203 bp 171–214 bp

TPOX 6–14 69–101 bp 217–249 bp 224–256 bp

VWA 11–22 126–170 bp 156–200 bp 127–171 bp

Other STRs

GATA132B04 10–14 99–115 bp

D22S445 10–16 110–130 bp N.A.

D16S2622 4–8 71–87 bp N.A.

* Sizes are listed without adenylation (add 1 base for +A form). 

N.A. = Not Available. 

Exhibit 2. PCR product sizes with newly designed primers for commonly 

used STR loci compared with commercially available primers used in 

multiplex sets for fluorescence-based assays. The red numbers indicate PCR 

product size ranges that exceed the recommended 140 bp mass spectrometry 

detection range.

N.A.
4

EXECUTIVE SUMMARY

using validated fluorescent multiplex

STR kits from ABI. GeneTrace generated STR results using their primer sets

for 9 STR loci (TH01, TPOX, CSF1PO,

D3S1358, D16S539, D8S1179, FGA,

DYS391, and D7S820) along with the

sex-typing marker amelogenin. These

experiments compared more than 700

genotypes (88 samples ✕ 8 loci; data

from D8S1179 and DYS391 were

not available from CDOJ). Although

results were not obtained for all possible samples using mass spectrometry,

researchers observed almost 100% correlation with the genotypes obtained

between the validated fluorescent STR

method and GeneTrace’s newly developed mass spectrometry technique,

demonstrating that the GeneTrace

method was reliable (see Results and

Discussion of STR Analysis by Mass

Spectrometry).

Multiplex STR analysis

To reduce analysis cost and sample

consumption and to meet the demands

of higher sample throughputs, PCR

amplification and detection of multiple

markers (multiplex STR analysis) has

become a standard technique in most

forensic DNA laboratories. STR multiplexing is most commonly performed

using spectrally distinguishable fluorescent tags and/or nonoverlapping

PCR product sizes. An example of an

STR multiplex produced from a commercially available kit is shown in

exhibit 3. Multiplex STR amplification in one or two PCR reactions

with fluorescently labeled primers

and measurement with gel or capillary

electrophoresis separation and laserinduced fluorescence detection is

becoming a standard method among

forensic laboratories for analysis of the

13 CODIS STR loci. The STR alleles

from these multiplexed PCR products

typically range in size from 100–350

bp with commercially available kits. 

Due to the limited DNA size constraints

of mass spectrometry, GeneTrace adopted a different approach to multiplex

analysis of multiple STR loci. Primers

 

D3S1358

D16S539 amelogenin

TH01

TPOX

CSF1PO

D7S820

GS350-ROX

Exhibit 3. Fluorescent multiplex STR result with AmpFlSTR® COfilerTM kit 

and ABI 310 Genetic Analyzer. The red peaks are an internal sizing standard 

(GS350-ROX). The DNA size window shown here is 90–350 bp. The allele 

calls for the seven loci in this multiplex are amelogenin: X,Y; D3S1358: 15, 17; 

TH01: 6, 7; TPOX: 8, 12; D16S539: 8, 12; D7S820: 9, 11; and CSF1PO: 12, 12.

90 120 150 180 210 240 270 300 330

6

Exhibit 4. Schematic of expected allele masses for a CSF1PO-TPOX-TH01 

(CTT) multiplex involving overlapping allele size ranges. All known alleles 

are fully distinguishable by mass with this interleaving approach. 

8,000 12,000 16,000 20,000 24,000 28,000

Mass (Da)

TPOX

THO1

7 8 9 10 11 12 13 14

10 11 12 13

12

6 7 8 9

3 5 6 7 8 8.3 9.3 10.3

CSF1PO

13.3

GeneTrace System

14 15

9 10 11
5

EXECUTIVE SUMMARY

are designed such that the PCR product size ranges overlap between multiple loci but have alleles that interleave

and are resolvable in the mass spectrometer (exhibit 4). As described above,

PCR primers are closer to the STR

repeat regions than those commonly

used with electrophoresis systems.

The high accuracy, precision, and 

resolution of this mass spectrometry

approach permits multiplexing STR

loci for a limited number of markers.

During the study, GeneTrace also

developed a TH01-TPOX-CSF1PO 

STR triplex (exhibit 5).

Single Nucleotide

Polymorphisms

Single nucleotide polymorphisms

(SNPs) represent another form of

DNA variation that is useful for

human identity testing. SNPs are the

most frequent form of DNA sequence

variation in the human genome and

are becoming increasingly popular

genetic markers for genome mapping

studies and medical diagnostics. SNPs

are typically biallelic with two possible

nucleotides (nt) or alleles at a particular site in the genome. Because SNPs

are less polymorphic (i.e., have fewer

alleles) than the currently used STR

markers, more SNP markers are

required to obtain the same level of

discrimination between samples.

Current estimates are that 30–50

unlinked SNPs will be required to

obtain the matching probabilities of

1 in ~100 billion as seen with the 

13 CODIS STRs.

The perceived value of SNPs for DNA

typing in a forensic setting include the

following:

◆ More rapid analysis.

◆ Cheaper costs.

◆ Simpler interpretation of results

because there are no stutter 

products.

◆ Improved ability to handle degraded DNA because of the possibility

of smaller PCR product sizes.

While it is doubtful that autosomal

SNPs will replace the current battery

of STRs used in forensic laboratories

in the near future, abundant mitochondrial and Y-chromosome SNP

markers exist and have already proven

useful as screening tools. These maternal (mitochondrial) and paternal (Y

chromosome) lineage markers are

effective in identifying missing persons

and war casualties and helping answer

historical questions such as whether

or not Thomas Jefferson fathered a

slave child.

The forensic DNA community already

has experience with applying SNP

markers as a screening process, which

can prove very helpful for excluding

suspects from crime scenes. Many

crime laboratories still use reverse dot

blot technology for analyzing the SNPs

from HLA-DQA1 and PolyMarker loci

with kits from ABI. In addition, mitochondrial DNA (mtDNA) sequencing

is currently performed in some forensic laboratories.

In the work performed on multiplex

SNP markers at GeneTrace, the

authors examined 10 polymorphic

sites within the mtDNA control region

and 20 Y-chromosome SNPs provided

by Dr. Peter Oefner and Dr. Peter

Underhill from Stanford University.

A multiplex SNP assay was developed

for 10 mtDNA SNP sites (exhibit 6).

Only limited work was performed on

the Y-chromosome SNPs due to the

premature termination of the work.

However, results demonstrated a malespecific 17-plex PCR of 17 different Y

SNP markers (exhibit 7).

Exhibit 5. Mass spectrum of an STR triplex involving TPOX, TH01, and 

CSF1PO. This sample is a mass spectrometry result using the interleaving allele 

approach schematically illustrated in exhibit 4. Multiplex PCR and multiplex 

primer extension with ddC termination were used to obtain this result.

10,000 12,000 14,000 16,000 18,000 20,000

Mass (Da)

22,000 24,000

primer

dimer

TPOX-8

Genotype

TPOX = 8,8

TH01 = 6,9.3

CSF = 12,12

CTT Multiplex

Signal

TH01-9.3

CSF1PO-12 TH01-6
6

EXECUTIVE SUMMARY

Conclusions and

Implications 

Time-of-flight mass spectrometry

offers a rapid, cost-effective alternative

for genotyping large numbers of samples. Each DNA sample can be accurately measured in a few seconds.

Due to the increased accuracy of mass

spectrometry, STR alleles can be reliably typed without comparison with

allelic ladders. Mass spectrometry

holds significant promise as a technology for high-throughput DNA

processing that will be valuable for

large-scale DNA database work.

In summary, the positive features of

mass spectrometry for STR analysis

include:

◆ Rapid results—STR typing at a

rate of seconds per sample.

◆ Accuracy—no allelic ladders.

◆ Direct DNA measurement—no 

fluorescent or radioactive labels.

◆ Automated sample preparation

and data collection.

◆ High-throughput capabilities

of thousands of samples daily 

per system.

◆ Flexibility—single nucleotide

polymorphism (SNP) assays can 

be run on the same instrument

platform.

This project demonstrates that both

STR and SNP analysis are reliably performed with GeneTrace’s mass spectrometry technology. Tests were done

on a large number of human DNA

markers of forensic interest. New

primer sets were developed for the

13 CODIS STR loci that may prove

useful in the future for situations in

which degraded DNA is present and

requires smaller amplicons to obtain

successful results. The possibility of

developing multiplexed SNP markers

also was explored, and a mtDNA 

10-plex assay and Y-chromosome, 

17-plex, male-specific PCR were

demonstrated. Both STR and SNP

areas appear promising for future

research. In another project, GeneTrace

recently demonstrated a sample

throughput of approximately 4,000

STR samples in a single day with a

single automated mass spectrometer.

Clearly, this is an improvement in the

analysis of DNA short tandem repeat

markers using time-of-flight mass

spectrometry.

Exhibit 6. Mass spectrum of SNP 10-plex assay for screening polymorphic 

sites in the mtDNA control region. The bottom panel shows the 10 SNP 

primers prior to the primer extension reaction. The top panel contains the 

multiplexed reaction products, each labeled with the observed extension 

product. The results for this K562 PCR product are (in order across HV1 and 

HV2): H16069 (G), H16129 (C), H16189 (A), H16224 (G), H16311 (A), 

H00073 (C), L00146 (T), H00152 (A), L00195 (T), and H00247 (C). SNP 

nucleotide results have been confirmed by sequencing. The primer sequences 

are listed in exhibit 24. The primer concentrations were 25 pmol MT5, 15 

pmol MT8’, 15 pmol MT10, 10 pmol MT3’, 20 pmol MT9, 25 pmol MT6, 

20 pmol MT2g, 27 pmol MT1, 35 pmol MT7, and 20 pmol MT4e. 

2,000 3,000 4,000 5,000 6,000 7,000

m/z

+ddA

+ddA

+ddA

+ddT +ddC

+ddG

+ddC +ddG

+ddC

+ddT

H16224

L00146

H00247 

H16189H00152H16311H16129H16069H10073H00195

primer

impurity

primers

alone

2 + ion

multiplex

extension

Signal
7

EXECUTIVE SUMMARY

Minutes

17-plex PCR 

Female sample (K562)

Male sample (AM209)

20 bp ladder

200 bp 300 bp 400 bp

Exhibit 7. Overlay of CE electropherograms demonstrating male-specific amplification with the 17-plex set of PCR 

primers. Note: The female sample (K562) failed to yield any peaks, illustrating that the PCR reaction is Y chromosomespecific. The PCR primers used are described in exhibit 27 with each locus-specific primer set at 0.4 pmole each. For the 

PCR reaction conditions, see exhibit 67 and details in the text. The 17 amplicons may be seen more clearly in exhibit 68. 

7.5 10.0
9

PROJECT DESCRIPTION

STR Grant

This project focused on the

development of a powerful new

technology for rapid and accurate analysis of DNA STR markers

using time-of-flight mass spectrometry.

GeneTrace Systems, Inc., collaborated

with the CDOJ DNA Laboratory in

Berkeley, California, primarily through

Dr. Steve Lee. This collaboration provided the study with the samples used

to verify the new GeneTrace technology, which was done by comparing the

mass spectrometry results with genotypes obtained using established and

validated methods run at CDOJ.

To accomplish the task of developing a

new mass spectrometry technology for

STR typing, five milestones were proposed in the original grant application,

which included the following:

◆ Redesign PCR primers for a 

number of commonly used STR

markers to produce smaller PCR

products that could be tested in

the mass spectrometer (exhibit 2).

◆ Demonstrate multiplexing capabilities to a level of 2 or 3 for detection with the TOF-MS method

(exhibits 4–5 and 8–10).

◆ Transfer the sample preparation

protocols from manual to a highly

parallel and automated pipetting

robot.

◆ Develop a large data set to confirm

the accuracy and reliability of this

method (exhibits 11–19).

◆ Automate and incorporate DNA

extraction techniques onto the

GeneTrace robots.

As described in the results section,

all milestones were met on time

except the final one regarding DNA

extraction. Two other companies,

Rosys and Qiagen, produced robotic

systems for DNA extraction after this

project began. Meanwhile, GeneTrace

remained focused on developing

other steps in DNA sample processing

since commercially available solutions

had already been developed, thereby

eliminating the need to include the

DNA extraction portion in this study.

Since this project began in June 1997,

a number of advances that impact the

ability to perform high-throughput

DNA typing have occurred in the

biotech field. In early 1998, ABI

released a dual 384-well PE9700

(“Viper”) thermal cycler, which makes

it possible to prepare 768 PCR samples

simultaneously. Beckman Instruments

also came to market with a 96-tip

Multimek pipetting robot. At the

beginning of this project, GeneTrace

used funds from this NIJ grant to purchase an MJ Research 384-well thermal cycler and a custom-built 96-tip

robotic pipettor on a CyberLab x-y-z

gantry. Both of these pieces of equipment were the state of the art at the

time but are now obsolete at GeneTrace

for routine operations and have been

replaced by the newer and more reliable products from Applied Biosystems

and Beckman Instruments.

Exhibit 8. Mass spectrum of an STR multiplex sample with nonoverlapping 

alleles. The two loci, TH01 and TPOX, were coamplified using the ddC 

termination approach. Note: The mass accuracy is improved for peaks closer 

to the calibration standard of 10,998 Da. 

10,000 12,000 14,000 16,000 18,000 20,000 22,000 24,000

TPOX-9 = 12,581 Da

TPOX-11 = 15,101 Da

TH01-7 = 17,336 Da

TH01-9.3 = 20,803 Da

Expected Mass

17,305 Da -31 Da

20,751 Da -52 Da

12,584 Da +3 Da

15,080 Da -21 Da

Observed Mass ∆ Mass

TPOX-9

TPOX-11

TH01-9.3

TH01-7

Signal

Mass (Da)
10

PROJECT DESCRIPTION

SNP Grant

The grant extension, which began in

August 1998, focused on the development of multiplexed SNP markers

from mtDNA and the Y chromosome.

Although the grant extension was terminated prematurely by GeneTrace

management in April 1999, portions

of the first four milestones were accomplished. The five milestones described

in the original grant extension included the following:

◆ Produce and test a set of 10 or

more SNP probes for mtDNA control region “hot spots” (exhibit 5).

◆ Develop software for multiplex

SNP analysis and data interpretation.

◆ Examine individual Y-chromosome

SNP markers.

◆ Develop multiplex PCR and multiplex SNP probes for Y-chromosome

SNP loci (exhibit 7).

◆ Determine the discriminatory

power for a set of Y-chromosome

markers by running ~300 samples

across 50 Y-chromosome SNP

markers.

The goal of the grant extension project

was to develop highly multiplexed SNP

assays that worked in a robust manner

with mass spectrometry and could be

genotyped in an automated fashion.

GeneTrace planned to select markers

with a high degree of discrimination to

aid in rapid screening of mitochondrial

DNA and Y-chromosome polymorphisms with the capability to handle

analysis of large databases of offender

DNA. At the time this proposal was

written (December 1997), GeneTrace

still intended to provide reagents and

instruments to large DNA service laboratories or to provide a DNA typing

service to the forensic DNA community.

The grant extension was prematurely

terminated due to a change in business

focus and a need to consolidate the

research efforts at GeneTrace.

Exhibit 9. Mass spectrum of STR multiplex mixture of TH01 and TPOX 

allelic ladders. The PCR products from the two loci differ by 120 Da. The 

ladders were reamplified from AmpF1STR® Green I kit materials. The TH01 

ladder ranges from 5 to 10 repeats with 9.3 included; the TPOX ladder ranges 

from 6 to 13 repeats with 13 not shown here.

14,000 16,000 18,000 20,000 22,000

TH01

5

TH01 Allelic Ladder

TPOX Allelic Ladder

TH01

6

TP0X

6

TP0X

7 TH01

7

TH01

8

TP0X

8

TP0X

9

TH01

9

TH01

9.3

TH01

10

TP0X

10 TP0X

11

TP0X

12

Signal

Mass (Da)

12,000 13,000 14,000 15,000 16,000

Exhibit 10. Mass spectrum of a multiplexed TH01 and TPOX sample 

showing interleaved amplicons that are only 85 Da apart 

TPOX 8

Mass (Da)

TPOX 11

TH01 6 TH01 7

∆m = 85 Da

Signal
11

PROJECT DESCRIPTION

ABI 310 Runs

January 5, 1999–January 8, 1999:

• Multiplex STR detection and analysis (COfilerTM kit amplifies 6 STRs + amelogenin simultaneously: TH01, TPOX, 

D7S820)

• ~54 hours of continuous operation (107 injections that included 88 samples, 9 allelic ladders, and reinjections of 

• ~2209 sec/sample or 316 sec/genotype (~5 min/genotype)

Mass Spectrometry Runs

Date STR Loci Tested

Number of

samples

Time Required for

Data Collection

Average Time

per Sample

Feb 11, 1998 TH01 88 + 8 controls 76 min for 96 samples ~50 sec/sample

May 6, 1998 TH01 6 samples 2.5 min for 6 samples ~25 sec/sample

October 1, 1998 TPOX, CSF1PO,

D3S1358, D7S820

72 + 8 controls

for each locus

60 min for 320 samples 11.3 sec/sample

Jan 12, 1999 TH01, TPOX, CSF1PO, 

D7S820

88 + 8 controls

for each locus

30 min for 384 samples 4.7 sec/sample

Jan 14, 1999 TH01, D16S539,

D3S1358, Amelogenin

88 + 8 controls

for each locus

37 min for 384 samples 5.8 sec/sample

Feb 12, 1999 D3S1358, FGA,

D8S1179, DYS391

88 + 8 controls

for each locus

41 min for 384 samples 6.4 sec/sample

March 26, 1999 TH01, TPOX, CSF1PO, 

D16S539

88 + 8 controls

for each locus

51 min for 384 samples 8.0 sec/sample

Exhibit 11. STR data collection times for CDOJ samples. Comparing ABI 310 run times with multiple mass spectrometry 

runs indicates that data collection speed increased almost 10-fold during 1998 due to a number of improvements in the 

GeneTrace process. Data collection speed is a combination of laser rate, total number of shots taken, sample cleanliness, 

and other factors. Overall, the mass spectrometry data collection speed is approximately two orders of magnitude faster 

than the ABI 310. Exhibits 12–19, 39, and 40 list the observed masses for these samples measured by GeneTrace’s mass 

spectrometry method.

CSF1PO, D3S1358, D16S539,

10 samples at ~30 min per injection)
Mass Spec

Exhibit 12. CDOJ CSF1PO results compared with ABI 310 and mass spectrometry methods

Position ABI 310

Allele 1

 (Da)

Allele 2 

(Da) Position ABI 310 Mass Spec

Allele 1

(Da)

Allele 2

(Da)

A1 11,12 11,12 27,823 29,060 A7 11,12 11,12 27,867 29,000

B1 10,12 10,12 26,520 29,033 B7 7,13 7,13 22,809 30,271

C1 7,10 7,10 22,917 26,684 C7 10,13 10,13 26,529 30,253

D1 10,13 10,13 26,586 30,344 D7 10,11 10,11 26,621 27,794

E1 8,12 8,12 24,077 29,075 E7 10,11 10,11 26,609 27,834

F1 8,10 8,10 24,107 26,639 F7 7,12 7,12 22,923 29,202

G1 12,13 12,13 29,144 30,373 G7 11,12 11,12 27,958 29,244

H1 11,12 11,12 27,812 29,018 H7 10,12 10,12 26,497 29,005

A2 12,12 12,12 29,033 A8 12,12 12,12 29,129

B2 10,10 10,10 26,594 B8 8,11 8,11 24,117 27,862

C2 10,10 10,10 26,550 C8 7,12 7,12 22,913 29,071

D2 10,11 10,11 26,609 27,823 D8 10,10 10,10 26,575

E2 10,12 10,12 26,620 29,129 E8 10,12 10,12 26,552 29,007

F2 10,10 10,10 26,584 F8 8,10 8,10 24,061 26,572

G2 11,12 11,12 27,862 29,080 G8 12,12 12,12 29,064

H2 10,10 10,10 26,605 H8 11,12 11,12 27,796 29,026

A3 12,12 12,12 28,987 A9 8,12 8,12 24,085 29,091

B3 10,13 10,13 26,614 30,380 B9 11,12 11,12 27,819 29,022

C3 10,10 10,10 26,618 C9 11,12 11,12 27,810 29,000

D3 11,11 11,11 27,823 D9 8,13 8,13 24,093 30,348

E3 11,12 11,12 27,817 28,998 E9 11,12 11,12 27,878 29,098

F3 11,12 11,12 27,871 29,093 F9 7,12 7,12 22,836 29,104

G3 10,11 10,11 26,616 27,812 G9 9,13 9,13 25,387 30,389

H3 11,12 11,12 27,849 29,024 H9 7,11 7,11 22,866 27,836

A4 8,10 8,10 24,048 26,499 A10 9,10 9,10 25,391 26,607

B4 11,13 11,13 27,941 30,412 B10 11,12 11,12 27,844 29,044

C4 11,11 11,11 27,873 C10 10,10 10,10 26,586

D4 10,11 10,11 26,638 27,794 D10 8,11 8,11 24,091 27,838

E4 7,10 7,10 22,835 26,553 E10 11,12 11,12 27,843 29,040

F4 10,14 10,14 26,592 31,565 F10 10,12 10,12 26,607 29,111

G4 12,14 12,14 29,113 31,625 G10 8,11 8,11 24,029 27,807

H4 9,10 9,10 25,360 26,582 H10 8,10 8,10 24,099 26,609

A5 10,10 10,10 26,590 A11 12,12 12,12 29,011

B5 7,11 7,11 22,818 27,838 B11 7,12 7,12 22,836 29,095

C5 10,10 10,10 26,628 C11 7,11 7,11 22,826 27,834

D5 10,11 10,11 26,647 27,925 D11 10,11 10,11 26,567 27,784

E5 11,12 11,12 27,847 29,031 E11 10,12 10,12 26,577 29,078

F5 10,10 10,10 26,601 F11 10,11 10,11 26,611 27,825

G5 10,12 10,12 26,603 29,113 G11 10,10 10,10 26,605

H5 11,13 11,13 27,856 30,389 H11 10,12 10,12 26,584 29,078

A6 11,11 11,11 27,832

B6 No data 10,12 26,654 29,180

C6 11,11 11,11 27,862

D6 11,12 11,12 27,818 29,079

E6 11,12 11,12 27,838 29,049

F6 8,10 8,10 24,206 26,739

G6 11,12 11,12 27,843 29,062

H6 11,11 11,11 27,773

SUMMARY:

Complete agreement observed among all 

genotypes.

12

PROJECT DESCRIPTION
13

PROJECT DESCRIPTION

Position ABI 310 Mass Spec

Allele 1

(Da)

Allele 2

(Da) Position ABI 310 Mass Spec

Allele 1

(Da)

Allele 2

(Da)

A1 8,10 8,10 17,914 20,431 A7 9,9 5,5 Gas-phase dimer

B1 6,8 6,8 15,376 17,897 B7 8,8 8,8 17,820

C1 9,11 9,11 19,137 21,663 C7 10,12 10,12 20,361 22,830

D1 11,12 11,12 21,562 22,801 D7 11,12 6,6 Gas-phase dimer

E1 9,10 9,10 19,131 20,374 E7 6,8 6,8 15,366 17,850

F1 9,12 9,12 19,106 22,889 F7 10,10 10,10 20,372

G1 8,8 8,8 17,838 G7 8,9 8,9 17,820 19,070

H1 8,8 8,8 17,825 H7 9,9 9,9 19,098

A2 8,8 8,8 17,837 A8 9,10 5,5 Gas-phase dimer

B2 8,8 8,8 17,841 B8 9,9 5,5 Gas-phase dimer

C2 8,9 8,9 17,860 19,101 C8 8,10 8,10 17,848 20,366

D2 8,9 8,9 17,785 19,049 D8 8,11 8,11 17,843 21,596

E2 8,11 8,11 17,829 21,577 E8 9,9 9,9 19,117

F2 8,12 8,12 17,834 22,816 F8 9,12 9,12 19,108 22,793

G2 7,10 7,10 16,525 20,257 G8 8,8 8,8 17,905

H2 8,9 8,9 17,870 19,171 H8 8,10 8,10 17,850 20,366

A3 8,10 8,10 17,844 20,366 A9 8,11 8,11 17,820 21,563

B3 9,10 9,10 19,063 20,320 B9 11,11 11,11 21,575

C3 9,11 9,11 19,178 21,676 C9 8,8 8,8 17,841

D3 7,10 7,10 16,626 20,370 D9 10,11 10,11 20,379 21,600

E3 9,11 9,11 19,146 21,665 E9 8,11 8,11 17,829 21,569

F3 8,10 8,10 17,839 20,357 F9 7,8 7,8 16,607 17,837

G3 8,8 8,8 17,853 G9 8,10 8,10 17,904 20,381

H3 6,9 6,9 15,313 19,070 H9 8,11 5,5 Gas-phase dimer

A4 6,8 6,8 15,359 17,895 A10 9,10 9,10 19,076 20,342

B4 11,11 11,11 21,579 B10 8,11 8,11 17,827 21,569

C4 8,9 8,9 17,865 19,126 C10 8,12 8,12 17,855 22,850

D4 8,9 8,9 17,839 19,088 D10 8,9 8,9 17,846 19,093

E4 6,9 6,9 15,289 19,070 E10 9,10 9,10 19,086 20,346

F4 9,11 5,5 Gas-phase dimer F10 9,12 9,12 19,065 22,801

G4 8,8 8,8 17,827 G10 11,12 11,12 21,554 22,795

H4 8,8 8,8 17,798 H10 8,11 8,11 17,841 21,584

A5 9,11 9,11 19,126 21,663 A11 6,9 6,9 15,299 19,076

B5 8,11 8,11 17,846 21,586 B11 8,9 8,9 17,851 19,095

C5 10,11 10,11 20,264 21,550 C11 9,11 9,11 19,120 21,592

D5 9,12 9,12 19,137 22,905 D11 8,8 8,8 17,829

E5 9,10 9,10 19,076 20,337 E11 11,11 11,11 21,569

F5 9,11 9,11 19,081 21,571 F11 11,11 11,11 21,548

G5 8,8 8,8 17,831 G11 11,11 11,11 21,579

H5 6,11 5,5 Gas-phase dimer H11 8,9 8,9 17,870 19,095

A6 8,11 5,5 Gas-phase dimer SUMMARY: Allele Number Avg. mass Std. Dev.

B6 No data 11,11 21,573 6 5 15,343 34.3

C6 8,8 8,8 17,844 7 3 16,586 53.7

D6 8,12 5,10 Gas-phase dimer 8 43 17,846 26.5

E6 8,12 8,12 17,831 22,816 9 31 19,102 33.1

F6 8,11 8,11 17,846 21,600 10 18 20,353 40.7

G6 9,11 9,11 19,070 21,565 11 24 21,590 37.9

H6 9,10 9,10 19,151 20,368 12 10 22,830 39.7

Exhibit 13. CDOJ TPOX results compared with ABI 310 and mass spectrometry methods
14

PROJECT DESCRIPTION

Position ABI 310 Mass Spec

Allele 1 

(Da)

Allele 2

 (Da) Position ABI 310 Mass Spec

Allele 1 

(Da)

Allele 2 

(Da)

A1 7,7 7,7 18,854 A7 7,8 7,8 18,877 20,104

B1 7,9.3 7,9.3 17,404 20,854 B7 8,9.3 8,9.3 20,128 22,337

C1 7,8 7,8 18,894 20,108 C7 8,9 8,9 20,193 21,415

D1 6,9 6,9 17,574 21,294 D7 6,9.3 6,9.3 16,167 20,876

E1 7,9.3 7,9.3 18,938 22,387 E7 9.3,9.3 9.3,9.3 22,349

F1 9,9 9,9 21,415 F7 9,9.3 9,9.3 21,417 22,345

G1 7,9 7,9 18,919 21,408 G7 7,8 7,8 18,911 20,147

H1 5,7 5,7 16,403 18,926 H7 9,9.3 9,9.3 21,400 22,277

A2 6,10 6,10 16,145 21,170 A8 8,8 8,8 20,091

B2 6,6 6,6 17,662 B8 7,9.3 7,9.3 18,938 22,391

C2 7,8 7,8 18,895 20,115 C8 7,9 7,9 17,436 19,957

D2 7,9 7,9 18,838 21,309 D8 7,7 7,7 18,944

E2 7,9.3 7,9.3 18,904 22,329 E8 7,7 7,7 17,376

F2 7,9 7,9 18,831 21,302 F8 7,9 7,9 17,387 19,891

G2 7,7 7,7 17,391 G8 9.3,9.3 9.3,9.3 22,263

H2 9,9.3 9,9.3 19,871 20,685 H8 7,9.3 7,9.3 18,829 22,267

A3 7,7 7,7 18,856 A9 9,9 9,9 19,907

B3 7,8 7,8 18,917 20,114 B9 7,7 7,7 18,938

C3 7,9.3 7,9.3 17,380 20,828 C9 9.3,9.3 9.3,9.3 22,351

D3 7,9 7,9 17,427 19,944 D9 7,8 7,8 18,845 20,097

E3 7,9.3 7,9.3 18,922 22,364 E9 8,9.3 8,9.3 18,684 20,790

F3 9,9.3 9,9.3 21,377 22,321 F9 6,9.3 6,9.3 17,622 22,287

G3 8,9.3 8,9.3 20,193 22,384 G9 7,9 7,9 17,380 19,885

H3 7,9.3 7,9.3 18,894 22,292 H9 6,7 6,7 17,677 18,926

A4 8,8 8,8 20,126 A10 7,8 7,8 17,389 18,652

B4 6,9.3 6,9.3 17,606 22,294 B10 9,9 9,9 21,377

C4 6,7 6,7 17,586 18,838 C10 7,9 7,9 17,294 19,812

D4 7,9.3 7,9.3 18,897 22,312 D10 7,8 7,8 17,277 18,547

E4 7,9 7,9 17,401 19,917 E10 6,7 6,7 17,570 18,819

F4 7,9 7,9 18,836 21,334 F10 8,8 8,8 18,640

G4 8,9.3 8,9.3 20,077 22,258 G10 6,7 6,7 17,644 18,906

H4 7,9.3 7,9.3 18,899 22,304 H10 8,9 8,9 18,643 19,902

A5 7,7 7,7 17,205 A11 6,7 6,7 17,540 18,782

B5 6,9 6,9 16,121 19,874 B11 6,8 6,8 17,682 20,191

C5 8,9.3 8,9.3 20,126 22,360 C11 7,7 7,7 18,927

D5 6,8 6,8 17,679 20,184 D11 7,7 7,7 17,477

E5 8,10 8,10 20,155 22,658 E11 6,6 6,6 17,596

F5 7,9.3 7,9.3 18,870 22,285 F11 7,8 7,8 17,400 18,656

G5 6,7 6,7 17,624 18,906 G11 8,8 8,8 18,710

H5 7,9 7,9 17,404 19,921 H11 6,7 6,7 17,600 18,895

A6 6,8 6,8 17,648 20,139

B6 7,7 7,7 18,910

C6 8,9.3 8,9.3 18,626 20,823

D6 8,8 8,8 20,147

E6 8,9.3 8,9.3 19,961 22,143

SUMMARY:

All 88 samples were in agreement.

F6 8,8 8,8 20,195

G6 7,9 7,9 18,858 21,383

H6 8,9 8,9 18,704 19,969

Exhibit 14. CDOJ TH01 STR results compared with ABI 310 and mass spectrometry methods. The shaded 

samples were run with a different primer set and have different masses.
PROJECT DESCRIPTION

Exhibit 15. CDOJ amelogenin results compared with ABI 310 and mass spectrometry methods

Position ABI 310 Mass Spec

Allele 1

(Da)

Allele 2

(Da) Position ABI 310 Mass Spec

Allele 1

(Da)

Allele 2 

(Da)

A1 X,Y X,Y 25,676 27,566 A7 X,Y X,Y 25,774 27,687

B1 X,Y X,Y 25,730 27,596 B7 X,Y X,Y 25,659 27,555

C1 X,Y X,Y 25,693 27,667 C7 X,Y X,Y 25,797 27,695

D1 X,Y X,Y 25,686 27,607 D7 X,Y X,Y 25,641 27,533

E1 X,Y X,Y 25,684 27,765 E7 X,Y X,Y 25,674 27,583

F1 X,Y X,Y 25,693 27,592 F7 X,Y X,Y 25,676 27,585

G1 X,Y X,Y 25,674 27,574 G7 X,Y X,Y 25,668 27,568

H1 X,Y X,Y 25,653 27,551 H7 X,Y X,Y 25,666 27,564

A2 X,Y X,Y 25,676 27,574 A8 X,Y X,Y 25,659 27,555

B2 X,Y X,Y 25,674 27,579 B8 X,Y X,Y 25,653 27,544

C2 X,Y X,Y 25,666 27,559 C8 X,Y X,Y 25,668 27,566

D2 X,Y X,Y 25,768 27,626 D8 X,Y X,Y 25,674 27,566

E2 X,Y X,Y 25,670 27,568 E8 X,Y X,Y 25,843 27,763

F2 X,Y X,Y 25,657 27,544 F8 X,Y X,Y 25,649 27,581

G2 X,Y X,Y 25,672 27,646 G8 X,Y X,Y 25,661 27,561

H2 X,Y X,Y 25,641 27,523 H8 X,Y X,Y 25,757 27,585

A3 X,Y X,Y 25,663 27,559 A9 X,Y X,Y 25,641 27,531

B3 X,Y X,Y 25,770 27,581 B9 X,Y X,Y 25,684 27,579

C3 X,Y X,Y 25,691 27,635 C9 X,X X,X 25,645

D3 X,Y X,Y 25,722 27,594 D9 X,Y X,Y 25,653 27,553

E3 X,Y X,Y 25,749 27,628 E9 X,Y X,Y 25,674 27,594

F3 X,Y X,Y 25,682 27,577 F9 X,Y X,Y 25,720 27,646

G3 X,Y X,Y 25,645 27,536 G9 X,Y X,Y 25,766 27,637

H3 X,Y No data H9 X,Y No data

A4 X,Y X,Y 25,666 27,553 A10 X,Y X,Y 25,697 27,618

B4 X,Y X,Y 25,726 27,637 B10 X,Y X,Y 25,684 27,590

C4 X,Y X,Y 25,736 27,583 C10 X,Y No data

D4 X,Y X,Y 25,782 27,670 D10 X,Y X,Y 25,657 27,564

E4 X,Y X,Y 25,701 27,789 E10 X,Y X,Y 25,657 27,549

F4 X,Y X,Y 25,674 27,585 F10 X,Y X,Y 25,653 27,551

G4 X,Y X,Y 25,599 27,572 G10 X,Y No data

H4 X,Y X,Y 25,682 27,579 H10 X,Y X,Y 25,722 27,654

A5 X,Y X,Y 25,816 27,719 A11 X,Y X,Y 25,676 27,572

B5 X,Y X,Y 25,672 27,570 B11 X,Y X,Y 25,655 27,549

C5 X,Y X,Y 25,680 27,583 C11 X,Y X,Y 25,666 27,577

D5 X,Y X,Y 25,688 27,661 D11 X,Y X,Y 25,618 27,464

E5 X,Y X,Y 25,643 27,523 E11 X,Y X,Y 25,697 27,676

F5 X,Y X,Y 25,666 27,572 F11 X,Y X,Y 25,653 27,557

G5 X,Y X,Y 25,778 27,672 G11 X,Y X,Y 25,661 27,553

H5 X,Y X,Y 25,738 27,568 H11 X,Y X,Y 25,643 27,540

A6 X,Y X,Y 25,695 27,587

B6 X,Y X,Y 25,649 27,523 SUMMARY: X allele Y allele

C6 X,Y X,Y 25,655 27,551 Count 82 81

D6 X,Y No data Average mass 25,690 Da 27,594 Da

E6 X,Y X,Y 25,720 27,605 Std. dev. 50.4 Da 59.6 Da

F6 X,Y X,Y 25,674 27,568 Eighty-two samples were in agreement.

G6 X,Y X,Y 25,881 27,745 “No data” resulted from mass spectrometry H6 X,Y No data in six samples.

15
16

PROJECT DESCRIPTION

Exhibit 16. CDOJ D3S1358 STR results compared with ABI 310 and mass spectrometry methods

Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da) Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da)

A1 17,17 17,17 28,182 A7 16,17 16,17 27,027 28,112

B1 16,16 16,16 26,967 B7 14,19 14,19 24,305 30,503

C1 15,17 15,17 25,668 28,139 C7 14,14 14,14 24,254

D1 12,17 12,17 21,820 27,991 D7 15,16 15,16 25,524 26,713

E1 15,15 15,15 25,741 E7 16,16 16,16 26,822

F1 16,16 16,16 26,762 F7 18,18 18,18 29,491

G1 14,15 14,15 24,342 25,501 G7 15,16 15,16 25,772 26,830

H1 15,16 15,16 25,802 26,941 H7 16,17 16,17 27,018 28,273

A2 15,17 15,17 25,638 28,134 A8 14,16 14,16 24,331 26,856

B2 16,17 16,17 26,795 27,889 B8 16,17 16,17 26,843 28,056

C2 15,16 15,16 25,661 26,869 C8 15,16 15,16 25,638 26,766

D2 13,16 13,16 23,171 27,091 D8 15,15 15,15 25,724

E2 14,17 14,17 24,352 28,126 E8 14,15 14,15 24,343 25,477

F2 15,17 15,17 25,549 28,091 F8 15,16 15,16 25,662 26,703

G2 15,17 15,17 25,641 28,119 G8 16,16 16,16 26,907

H2 14,15 14,15 24,311 25,507 H8 15,15 15,15 25,618

A3 17,17 17,17 28,184 A9 14,16 14,16 24,323 26,832

B3 18,18 18,18 29,384 B9 15,16 15,16 25,537 26,739

C3 15,15 15,15 25,645 C9 15,15 15,15 25,499

D3 15,15 15,15 25,645 D9 15,15 15,15 25,522

E3 16,17 16,17 26,967 27,945 E9 14,15 14,15 24,440 25,459

F3 15,16 15,16 25,573 26,790 F9 16,17 16,17 26,745 28,021

G3 17,18 17,18 28,025 29,302 G9 17,18 17,18 28,372 29,315

H3 14,15 14,15 24,360 25,530 H9 15,16 15,16 25,761 26,777

A4 17,17 17,17 28,001 A10 16,16 16,16 26,913

B4 15,16 15,16 25,518 26,811 B10 15,18 15,18 25,539 29,347

C4 15,16 15,16 25,705 26,722 C10 15,16 15,16 25,477 26,741

D4 15,17 15,17 25,589 28,110 D10 14,15 14,15 24,214 25,341

E4 16,17 16,17 27,035 28,023 E10 14,15 14,15 24,252 25,507

F4 14,16 14,16 24,374 26,886 F10 16,17 16,17 26,871 28,139

G4 14,17 14,17 24,342 28,115 G10 16,17 16,17 27,033 27,934

H4 15,18 15,18 25,655 29,378 H10 15,16 15,16 25,716 26,715

A5 15,16 15,16 25,727 27,110 A11 15,18 15,18 25,545 29,293

B5 16,17 16,17 26,988 28,300 B11 15,15.2 15,15.2 25,734 26,379

C5 15,15 15,15 25,684 C11 16,17 16,17 26,937 27,845

D5 14,16 14,16 24,520 26,941 D11 15,16 15,16 25,544 26,672

E5 15,17 15,17 25,678 28,132 E11 16,16 16,16 26,899

F5 16,16 16,16 26,813 F11 15,16 15,16 25,706 26,678

G5 9,17 9,17 18,092 28,285 G11 16,16 16,16 26,841

H5 17,17 17,17 27,893 H11 16,17 16,17 26,881 28,246

A6 16,17 16,17 26,999 28,178

B6 15,17 15,17 25,645 28,182 SUMMARY:

C6 15,16 15,16 25,681 26,710

D6 15,15 15,15 25,833 All 88 samples were in agreement.

ABI 310 CE electropherogram of boxed data

is shown in exhibit 74.

E6 14,16 14,16 24,301 26,816

F6 14,14 14,14 24,437

G6 14,15 14,15 24,425 25,524

H6 15,15 15,15 25,523
17

PROJECT DESCRIPTION

Exhibit 17. CDOJ D16S539 STR results compared with ABI 310 and mass spectrometry methods

Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da) Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da) 

A1 11,12 11,12 26,862 28,095 A7 11,11 11,11 26,873

B1 9,14 9,14 24,421 30,665 B7 9,10 9,10 24,338 25,595

C1 9,11 9,11 24,360 26,888 C7 11,13 11,13 26,894 29,471

D1 10,11 10,11 25,622 26,831 D7 11,11 11,11 26,879

E1 11,13 11,13 26,903 29,367 E7 9,12 9,12 24,425 28,200

F1 10,11 10,11 25,543 26,830 F7 9,12 9,12 24,368 28,160

G1 11,12 11,12 26,852 28,070 G7 11,12 11,12 26,997 28,145

H1 8,9 8,9 23,157 24,360 H7 12,12 12,12 28,104

A2 9,11 9,11 24,342 26,805 A8 9,13 9,13 24,360 29,362

B2 12,13 12,13 28,191 29,323 B8 9,9 9,9 24,394

C2 11,11 11,11 26,907 C8 9,13 9,13 24,352 29,369

D2 13,13 13,13 29,445 D8 11,11 11,11 26,933

E2 12,12 12,12 28,128 E8 8,12 8,12 23,332 28,486

F2 12,13 12,13 28,202 29,436 F8 11,12 11,12 26,937 28,128

G2 10,11 10,11 25,638 26,869 G8 9,13 9,13 24,336 29,351

H2 10,11 10,11 25,576 26,864 H8 11,14 11,14 26,864 30,573

A3 9,12 9,12 24,340 28,106 A9 11,13 11,13 26,869 29,378

B3 9,12 9,12 24,356 28,117 B9 11,12 11,12 26,888 28,078

C3 12,13 12,13 28,141 29,483 C9 12,12 12,12 28,106

D3 9,9 9,9 24,417 D9 11,13 11,13 26,849 29,342

E3 11,13 11,13 27,014 29,603 E9 11,13 11,13 26,899 29,396

F3 11,11 11,11 26,899 F9 12,15 12,15 28,115 31,845

G3 9,11 9,11 24,376 26,931 G9 11,13 11,13 26,886 29,389

H3 8,10 8,10 23,161 25,649 H9 11,13 11,13 26,888 29,387

A4 9,11 9,11 24,427 26,935 A10 9,11 9,11 24,423 26,920

B4 9,12 9,12 24,480 28,248 B10 9,12 9,12 24,449 28,126

C4 13,13 13,13 29,318 C10 12,13 12,13 28,302 29,498

D4 11,12 No data D10 8,9 8,9 23,072 24,317

E4 13,13 13,13 29,389 E10 9,13 9,13 24,376 29,402

F4 9,11 9,11 24,374 26,894 F10 11,13 11,13 26,856 29,436

G4 13,14 No data G10 9,9 9,9 24,358

H4 11,13 11,13 26,837 29,336 H10 10,13 10,13 25,653 29,441

A5 12,12 12,12 28,117 A11 9,12 9,12 24,427 28,128

B5 8,9 8,9 23,181 24,417 B11 11,12 11,12 26,862 28,043

C5 10,11 10,11 25,670 27,007 C11 11,12 No data

D5 12,12 12,12 28,265 D11 11,11 11,11 26,890

E5 9,11 9,11 24,482 26,984 E11 11,13 11,13 26,928 29,619

F5 9,11 9,11 24,329 26,854 F11 9,10 No data

G5 10,12 10,12 25,688 28,222 G11 10,12 No data

H5 11,12 11,12 26,918 28,041 H11 11,11 11,11 26,894

A6 9,11 9,11 24,372 26,888

B6 No data 9,12 24,405 28,208 SUMMARY:

C6 9,13 9,13 24,380 29,398 Eighty-one samples were in agreement.

D6 9,13 9,13 24,465 29,416 “No data” resulted from mass spectrometry in five samples. 

E6 12,12 12,12 28,170 “No data” resulted from ABI 310 in one sample.

F6 13,13 13,13 29,329 Gas-phase trimer caused an error in one sample. 

G6 11,13 11,13 26,867 29,371

H6 11,11 9,9 Gas-phase trimer
18

PROJECT DESCRIPTION

Exhibit 18. CDOJ D7S820 STR results compared with ABI 310 and mass spectrometry methods

Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da) Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da)

A1 11,13 11,13 20,322 22,807 A7 10,12 10,12 19,097 21,621

B1 10,11 10,11 19,081 20,333 B7 11,12 11,12 20,545 21,592

C1 8,11 8,11 16,641 20,379 C7 8 ,10 10,10 19,360

D1 9,10 9,10 17,857 19,131 D7 11,12 11,12 20,383 21,646

E1 8 ,10 10,10 19,072 E7 9,12 9,12 17,841 21,583

F1 10,12 10,12 19,088 21,594 F7 8, 9 8,8 16,631

G1 8,10 8,10 16,643 19,174 G7 11,12 11,12 20,374 21,575

H1 8,10 8,10 16,616 19,088 H7 11,12 11,12 20,366 21,581

A2 8,11 8,11 16,646 20,448 A8 10,12 10,12 19,189 21,703

B2 10,11 10,11 19,169 20,381 B8 10,11 10,11 19,265 20,400

C2 8,8 8,8 16,534 C8 10,10 10,10 19,095

D2 10,10 10,10 19,189 D8 9,10 9,10 17,848 19,093

E2 8,8 8,8 16,641 E8 8 ,10 10,10 19,169

F2 8,8 8,8 16,616 F8 8,9 No data Double null alleles?

G2 8 ,10 10,10 19,101 G8 9,12 9,12 17,949 21,745

H2 8,9 8,9 16,550 17,834 H8 8 ,10 10,10 19,187

A3 11,12 11,12 20,372 21,569 A9 8 ,11 11,11 20,571

B3 10,11 10,11 19,104 20,366 B9 8,8 8,8 16,534

C3 8,12 8,12 16,532 21,548 C9 10,10 10,10 19,169

D3 12,13 12,13 21,560 22,789 D9 7,8 7,8 15,383 16,638

E3 10,12 10,12 19,187 21,695 E9 9,10 9,10 17,844 19,090

F3 10,11 10,11 19,198 20,381 F9 10,12 10,12 19,395 21,866

G3 8 ,11 11,11 20,374 G9 8,10 8,10 16,651 19,201

H3 8 ,10 10,10 19,097 H9 8,11 8,11 16,826 20,363

A4 9,9 9,9 17,848 17,848 A10 12,13 12,13 21,586 22,820

B4 11,11 11,11 20,528 B10 11,12 11,12 20,387 21,586

C4 8,12 8,12 16,639 21,667 C10 8 ,10 10,10 19,330

D4 8,9 8,9 16,636 17,860 D10 8,11 8,11 16,554 20,342

E4 9 ,11 11,11 20,392 E10 10,11 10,11 19,086 20,348

F4 10,11 10,11 19,185 20,396 F10 9,10 9,10 17,851 19,097

G4 10,10 10,10 19,101 G10 9 ,10 10,10 19,398

H4 10 ,12 12,12 21,579 H10 10,11 10,11 19,382 20,377

A5 9,10 9,10 17,836 19,083 A11 10,10 10,10 19,254

B5 11,12 11,12 20,631 21,678 B11 10,11 10,11 19,093 20,353

C5 11,12 11,12 20,363 21,573 C11 9,10 9,10 17,886 19,187

D5 10,10 10,10 19,088 D11 8,11 8,11 16,643 20,519

E5 9,11 9,11 17,851 20,372 E11 10,10 10,10 19,252

F5 8,11 8,11 16,634 20,377 F11 9,12 9,12 17,831 21,569

G5 11,12 11,12 20,590 21,787 G11 10,12 10,12 19,142 21,653

H5 10,10 10,10 19,322 H11 10,12 10,12 19,160 21,883

A6 8,10 8,10 16,556 19,074

B6 8, 9 8,8 16,643

C6 8 ,11 11,11 20,374 SUMMARY:

D6 8,10 8,10 16,614 19,090 Seventy samples were in agreement.

E6 8 ,9 9,9 17,851 “No data” resulted from mass spectrometry 

(null alleles?) in one sample. F6 8,8 8,8 16,831

G6 8 ,10 10,10 19,155 Seventeen samples disagreed because of null alleles.

H6 9,9 9,9 18,169
19

PROJECT DESCRIPTION

Fifty-three samples were in agreement.

Exhibit 19. CDOJ FGA STR results compared with ABI 310 and mass spectrometry methods

Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da) Position ABI 310 Mass Spec Allele 1 (Da) Allele 2 (Da)

A1 20,21 No data A7 23,24 24,24 40,518 Not resolved

B1 20,23 20,23 35,987 39,547 B7 22,25 22,25 38,290 41,811

C1 23,25 23,25 39,390 41,917 C7 24,25 24,24 41,079 Not resolved

D1 19,23 19,23 34,858 39,547 D7 22,22 21,21 37,930

E1 23,24 23,23 40,317 Not resolved E7 24,27 24,27 40,680 44,130

F1 18.2,26 18.2,26 34,066 43,221 F7 22,22 22,22 38,295

G1 21,21 21,21 37,011 G7 22,23 22,22 38,582 Not resolved

H1 No data 23,23 39,555 H7 21,26 21,26 37,148 43,116

A2 22,23 23,23 39,446 Not resolved A8 OL?,24 16,24 31,415 40,688

B2 21,22 21,21 37,570 Not resolved B8 22,24 22,24 38,226 40,874

C2 21,25 21,25 37,146 41944 C8 19.2,25 19.2,25 35,193 41,718

D2 19,19.2 19,19 34,699 Not resolved D8 24,25 24,24 41,184 Not resolved

E2 23,27 23,26 39,310 43958 E8 18.2,24 18,23 33,960 40,247

F2 19,25 19,25 34,667 41575 F8 23,25 23,25 39,482 41,914

G2 22,22 22,22 38,554 G8 25,25 24,24 41,668

H2 22,25 22,25 38,244 41,861 H8 21,23 21,23 37,113 39632

A3 22,24 22,24 38,110 40,604 A9 19,25 19,25 34,631 41,827

B3 30.2, OL? 30,30 48,114 B9 20,24 20,24 35,888 40,688

C3 21,24 21,24 37,158 40,777 C9 20,24 19,23 35,667 40,312

D3 23,23 23,23 39,475 D9 21,24 21,24 36,973 40,573

E3 19,23 19,23 34,769 39,547 E9 21,25 21,24 36,921 41,549

F3 23,24 23,23 39,852 Not resolved F9 25,26 25,25 42,218 Not resolved

G3 22,23 22,22 38,582 Not resolved G9 20,21 21,21 36,901 Not resolved

H3 18.2,23 18.2,23 34,138 39,423 H9 18.2,25 18.2,25 34,111 41,774

A4 22,23 22,22 39,056 Not resolved A10 24,28 24,28 40,706 45,467

B4 19,26 19,26 34,578 42761 B10 21,24 21,24 37,018 40,636

C4 24,24 24,24 40,649 C10 18,23 18,23 33,560 39,490

D4 26,27 No data D10 22,23 22,22 38,537 Not resolved

E4 22,22 22,22 38,442 E10 18,23 18,23 33,292 39,289

F4 22,23 22,22 39,081 Not resolved F10 19,22 19,22 34,829 38,402

G4 23,24 23,23 39,741 Not resolved G10 22,24 22,24 38,216 40,581

H4 23,23 23,23 39,562 H10 20,26 20,26 36,127 43,407

A5 20,22 20,22 35,856 38,364 A11 24,26 24,26 40,659 43,043

B5 23,23 22,22 39,017 B11 23,28 23,28 39,493 45,376

C5 22,23 22,22 38,659 Not resolved C11 24,25 24,24 40,869 Not resolved

D5 23,26 22,25 39,233 42,879 D11 22,25 22,25 38,361 41,989

E5 22,23 22,22 38,554 Not resolved E11 25,26 25,25 42,333 Not resolved

F5 19,25 19,25 34,795 41,994 F11 22,24 22,24 38,209 40,560

G5 21,24 21,24 37,366 40,853 G11 23,25 23,25 39,511 41,909

H5 21,22 21,21 37,565 Not resolved H11 23,26 23,26 39,408 42,965

A6 22,24 22,24 38,155 40,570 SUMMARY:

B6 24,27 24,27 40,612 44,288

C6 21,25 21,25 37,136 41,997 “No data” resulted from mass spectrometry in two samples.

D6 20,23 20,23 35,972 39,477 “No data” resulted from ABI 310 in one sample.

E6 22,23 22,22 38,644 Not resolved One sample was too large for detection. 

F6 25,28 25,27 41,737 45,299 Nine samples resulted in wrong calls from 

poor calibration.

G6 18.2,24 18.2,24 34,387 40,869 Twenty-two samples resulted in wrong calls from 

H6 21,23 21,23 37,113 39,534 poor resolution.
21

SCOPE AND METHODOLOGY

Both the STR and the SNP genotyping assays used in this projectinvolve the same fundamental

(proprietary) sample preparation chemistry. This chemistry was important for

salt reduction/removal prior to the mass

spectrometric analysis and was automated on a 96-tip robotic workstation.

A biotinylated, cleavable oligonucleotide was used as a primer in each

assay and was incorporated through

standard DNA amplification (i.e., PCR)

methodologies into the final product,

which was measured in the mass spectrometer. This process was covered by

U.S. Patent 5,700,642, which was issued

in December 1997, and is described in

more detail in U.S. Patent 6,090,588

(Butler et al., 2000). The STR assay is

schematically illustrated in exhibit 20

and involves a PCR amplification step

where one of the primers is replaced by

the GeneTrace cleavable primer. The

biotinylated PCR product was then captured on streptavidin-coated magnetic

beads for post-PCR sample cleanup 

and salt removal followed by mass 

spectrometry analysis.

The biology portion of the SNP assay,

on the other hand, involves a three-step

process: (1) PCR amplification, (2)

phosphatase removal of nucleotides,

and (3) primer extension, using the

GeneTrace cleavable primer, with

dideoxynucleotides for single-base

addition of the nucleotide(s) complementary to the one(s) at the SNP site

(Li et al., 1999). The SNP assay is

illustrated in exhibit 21.

Simultaneous analysis of multiple

SNP markers (i.e., multiplexing) is

possible by simply putting the cleavage sites at different positions in the

various primers so they do not overlap on a mass scale. Also important to

both genotyping assays is proprietary

calling software that was developed

(and evolved) during the course of

this work. A number of STR and SNP

markers were developed and tested

with a variety of human DNA samples

as part of this project to demonstrate

the feasibility of this mass spectrometry approach.

B

B

PCR

(incorporates GTS primer)

Solid-Phase Purification

(reduces salt)

Cleavage

(reduces size and releases product)

Mass Spec Analysis

25,625 Da

Genotype Samples

(using mass-to-allele conversion file)

12 AATG repeats

GTS primer

Regular primer

Repeat region

Biotin

Cleavable base

Streptavidin-coated

magnetic bead

CallSTR

Program

Exhibit 20. Schematic of the STR assay using the GeneTrace cleavable 

primer technology
22

SCOPE AND METHODOLOGY

Assay Development

and Primer Testing

Primer design

Primers were initially designed for

each STR locus using Gene Runner

software (Hastings Software, Inc.,

Hastings, NY) and then more recently with Primer 3 version 0.2 from 

the World Wide Web (http://wwwgenome.wi.mit.edu/cgi-bin/primer/

primer3_www.cgi) (Rozen and

Skaletsky, 1998). Multiplex PCR

primers for the multiplex SNP work

were designed with a UNIX version 

of Primer 3 (release 0.6) adapted at

GeneTrace to utilize a mispriming

library and Perl scripts for input of

sequences and export of primer

information. 

DNA sequence information was

obtained from GenBank (http://www.

ncbi.nlm.nih.gov) and STRBase (http://

www.cstl.nist.gov/biotech/strbase) for

the STR loci and mtDNA and from Dr.

Peter Underhill of Stanford University

for the Y-chromosome SNPs. These

sequences served as the reference

sequence for primer design and, in

the case of STRs, the calibrating mass

for the genotyping software (see below).

When possible, primers were placed

close to the repeat region to make the

PCR product size ranges under 120 bp

to improve the sensitivity and resolution in the mass spectrometer (exhibit 2). Previously published primers

were used in the case of amelogenin

(Sullivan et al., 1993), D3S1358 

(Li et al., 1993), CD4 (Hammond

et al., 1994), and VWA (Fregeau and

Fourney, 1993) because their PCR

product sizes were analyzable in the

mass spectrometer or the amplicons

could be reduced in size following 

the PCR step (see below). Later,

D3S1358 experiments were performed

with primers that produced smaller

products after sequence information

became available for that particular

STR locus (exhibit 22).

Primer synthesis

Unmodified primers were purchased

from Biosource/Keystone (Foster City,

CA) or Operon Technologies

(Alameda, CA) or synthesized inhouse using standard solid-phase

phosphoramidite chemistry. The

GeneTrace cleavable primers were synthesized in-house using a proprietary

phosphoramidite that was incorporated near the 3’ end of the oligonucleotide along with a biotin attached at

the 5’ end. Primers were quality control tested via mass spectrometry prior

PCR Amplification With Regular Primers

(G/A)

Single Base Primer Extension

B ddC/ddT

Solid-Phase Purification

(reduces salt)

Biotin Cleavable base

Streptavidin-coated

magnetic beads

Cleavage

(reduces size)

ddC/ddT

Mass Spec Analysis

Unextended

primer

+ddC

+ddT

B

B ddC/ddT

mass = SNP base(s) present

ddC

ddT 288 Da

ddA 297 Da

ddG 313 Da

Phosphatase Treatment

(removes dNTPs)

Addition of polymerase, ddNTPs,

and GTS primer

Exhibit 21. Schematic of the SNP assay using the GeneTrace cleavable 

primer technology

273 Da

Expected Mass Differences
to further testing to confirm proper

synthesis and to determine the presence or absence of failure products.

Synthesis failure products (i.e., n-1,

n-2, etc.) can especially interfere with

multiplex SNP analysis. The cleavable

base is stable during primer synthesis

and PCR amplification. Comparisons

of regular primers with cleavable

primers containing the same base

sequence showed no significant difference, indicating that the primer

annealing is not compromised by the

cleavable base.

Methods for STR product

size reduction

It was discovered early in the study

that the PCR primer opposite the

biotinylated cleavable primer could be

moved into the repeat region as much

as two full repeat units to reduce the

overall size without severely compromising the PCR reaction. For the

cleavable primer, the cleavable base

was typically placed in the second or

third position from the 3’ end of the

primer in order to remove as much of

the modified primer as possible. Thus,

the cleavage step reduces the overall

PCR product size by the length of the

cleavable primer minus two or three

nucleotides. Typically, this size reduction is approximately 20 bases.

The portion of the DNA product on the

other side of the repeat region from the

cleavable primer was removed in one of

two possible ways: using a restriction

enzyme (Monforte et al., 1999) or performing a nested linear amplification

with a terminating nucleotide (Braun 

et al., 1997a and 1997b), such as

dideoxynucleotide (ddN). These methods work only for particular situations

(see Results and Discussion). Almost

all singleplex STR work was performed

without either of these product size

reduction methods. However, these size

reduction methods played a role in the

multiplex STR work.

Multiplex design

STR multiplexes were designed by

construction of virtual allelic ladders

or “mass simuplexes” that involved the

predicted mass of all known alleles for 

a particular locus. STR markers were

then interleaved based on mass with all

alleles between loci being distinguishable (exhibit 4). STR multiplexes work

best if alleles are below 20,000–25,000

Daltons (Da) in mass due to the

improved sensitivity and resolution

that is obtainable in the mass spectrometer. As previously described in

the section on size reduction, a restriction enzyme or a ddN terminator may

be used to shorten the STR allele sizes.

For multiplex design, locating a restriction enzyme with cut sites common to

all STR loci involved in the multiplex

complicates the design process and

limits the choice of possible marker

combinations. The use of a common

dideoxynucleotide terminator is much

easier. For example, with the STR loci

CSF1PO, TPOX, and TH01, a multiplex was developed using a dideoxycytosine (ddC) terminator and primer

extension along the AATG strand

(exhibits 4 and 5).

SNP multiplexes were designed by calculating possible postcleavage primer

and extension product masses. Multiply

charged ions were abundant in the mass

range of 1,500–7,000 Da in SNP multiplex analyses, which were avoided for

the most part by calculating interfering doubly charged and triply charged

ions. The cleavage sites for candidate

multiplex SNP primers were chosen for

the least amount of overlap between

singly and multiply charged ions

(exhibits 23).

23

SCOPE AND METHODOLOGY

(A) Previously Published Primers: PCR product size = 127 bp for 15 repeats

(B) Newly Designed Primers: PCR product size = 100 bp for 15 repeats

Exhibit 22. D3S1358 sequence with PCR primer locations. This STR 

sequence was not publicly available in GenBank at the start of this project but 

was obtained as part of this work. The forward primer is shown in blue and the 

reverse primer in red, with the GATA repeat region shaded grey. Section (A) 

shows the primer locations compared with the repeat region for a primer pair 

originally published by Li and colleagues (1993). Section (B) shows the 

primers designed as part of this project and their positions relative to the repeat 

region. The overall size of PCR products was reduced by 27 bp (more than 

8,000 Da) compared with an amplicon generated from the previously published 

primer set.
24

SCOPE AND METHODOLOGY

Human DNA samples used

Human genomic DNA samples representing several ethnic groups (AfricanAmerican, European, and Asian) were

purchased from Bios Laboratories (New

Haven, CT) for the initial studies. K562

cell line DNA (Promega) was used as a

control sample since the genotypes for

this cell line were reported in most of

the STR loci (GenePrintTM STR Systems

Technical Manual, 1995).

Allelic ladders were reamplified from a

1:1000 dilution of each of the allelic

ladders supplied in fluorescent STR

kits from ABI using the PCR conditions

listed below and the primers shown in

exhibit 24. The ABI kits included allelic

1,580

1,790

2,785

3,179

3,740

4,355

4,957

5,375

5,891

6,500

Name Singly Charged Ions Doubly Charged Ions Triply Charged Ions

ddC ddT ddA ddG ddC ddT ddA ddG

Primer

Mass

Primer

Mass

Primer

Mass

ddC ddT ddA ddG

MT5 1,877 1,893 790 939 947 527 626 631

MT8’ 2,063 2,078 895 1,032 1,039 597 688 693

MT10 3,058 3,073 1,393 1,529 1,537 928 1,019 1,024

MT3’ 3,476 3,492 1,590 1,738 1,746 1,060 1,159 1,164

MT9 4,037 4,053 1,870 2,019 2,027 1,247 1,346 1,351

MT6 4,652 4,668 2,178 2,326 2,334 1,452 1,551 1,556

MT2g 5,230 5,245 2,479 2,615 2,623 1,652 1,743 1,748

MT1 5,672 5,688 2,688 2,836 2,844 1,792 1,891 1,896

MT7 6,164 6,179 2,946 3,082 3,090 1,964 2,055 2,060

MT4e 6,773 6,788 3,250 3,387 3,394 2,167 2,258 2,263

Exhibit 23. Expected mass-to-charge ratios for various ions in the mtDNA 10-plex assay

STR Locus Primer Sequences for GTS Mass Spec Analysis

Primer

Name

Amelogenin 5’-b-CCCTGGGCTCTGTAAAGAATAG(T)G-3’

5’-ATCAGAGCTTAAACTGGGAAGCTG-3’

AMEL-F

AMEL-R

CD4 5’-b-TTGGAGTCGCAAGCTGAAC(T)AGC-3’

5’-GCCTGAGTGACAGAGTGAGAACC-3’

CD4-F

CD4-R

CSF1PO 5’-ACAGTAACTGCCTTCATAGA(T)AG-3’

5’-b-GTGTCAGACCCTGTTCTAAGTA-3’

CSF-F3

CSF-R3

D3S1358 5’-b-CAGAGCAAGACCCTGTC(T)CAT-3’

5’-TCAACAGAGGCTTGCATGTAT-3’

D3-F2

D3-R2

D5S818 5’-b-CTCTTTGGTATCCTTATGTAATA(T)T-3’

5’-ATCTGTATCCTTATTTATACCTCTATCTA-3’

D5-F

D5-R

D7S820 5’-b-TGTCATAGTTTAGAACGAAC(T)AAC-3’

5’-AAAAACTATCAATCTGTCTATCTATC-3’

D7-F

D7-R

D8S1179 5’-b-TTTGTATTTCATGTGTACATTCGTA(T)C-3’

5’-ACCTATCCTGTAGATTATTTTCACTGTG-3’

D8-F

D8-R

D13S317 5’-b-CCCATCTAACGCCTATCTGTA(T)T-3’

5’-GCCCAAAAAGACAGACAGAAAG –3’

D13-F

D13-R2

D16S539 5’-b-ATACAGACAGACAGACAGG(T)G-3’

5’-GCATGTATCTATCATCCATCTCT-3’

D16-F4

D16-R4

D18S51 5’-TGAGTGACAAATTGAGACCTT-3’

5’-b-GTCTTACAATAACAGTTGCTACTA(T)T-3’

D18-F

D18-R

D21S11 5’-b-CCCAAGTGAATTGCCTTC(T)A-3’

5’-GTAGATAGACTGGATAGATAGACGATAGA-3’

D21-F

D21-R

F13A1 5’-b-CAGAGCAAGACTTCATC(T)G-3’

5’-TCATTTTAGTGCATGTTC-3’

F13A1-F

F13A1-R

FES/FPS 5’-b-TTAGGAGACAAGGATAGCAGT(T)C-3’

5’-GCGAAAGAATGAGACTACATCT-3’

FES-F2

FES-R2

FGA 5’-b-AAAATTAGGCATATTTACAAGCTAG(T)T-3’

5’-TCTGTAATTGCCAGCAAAAAAGAAA-3’

FGA-F

FGA-R

HPRTB 5’-b-GTCTCCATCTTTGTCTCTATCTCTATC(T)G-3’

5’-GAGAAGGGCATGAATTTGCTTT-3’

HPRTB-F

HPRTB-R

5’-b-CTGACCAAGGATAGTGGGATA(T)AG-3’

5’-GGTAACTGAGCGAGACTGTGTCT-3’

LPL-F

LPL-R

TH01 5’-CCTGTTCCTCCCTTATTTCCC-3’

5’-b-GGGAACACAGACTCCATGG(T)G-3’

TH01-F

TH01-R

TPOX 5’-b-CTTAGGGAACCCTCACTGAA(T)G-3’ TPOX-F

Exhibit 24. Primer sequences designed for STR markers tested by mass spectrometry. For the PCR product size 

produced with these primers, see exhibit 2. Note: “b” is listed for biotin; parentheses ( ) indicate the cleavable base. 

STR Locus Primer Sequences for GTS Mass Spec Analysis

Primer

Name

LPL

VWA 5’-b-CCCTAGTGGATGATAAGAATAATCAGTATG-3’

5’-b-GGACAGATGATAAATACATAGGATGGA(T)GG-3’

5’-b-GTCCTTGTCAGCGTTTATTTGC-3’ TPOX-R

VWA-F

VWA-R
25

SCOPE AND METHODOLOGY

ladders for the following STR loci:

AmpF1STR® Green I (CSF1PO, TPOX,

TH01, amelogenin), AmpF1STR® Blue

(D3S1358, VWA, FGA), AmpF1STR®

Green II (amelogenin, D8S1179,

D21S11, D18S51), AmpF1STR® Yellow

(D5S818, D13S317, D7S820), and

AmpF1STR® COfilerTM (amelogenin,

TH01, TPOX, CSF1PO, D3S1358,

D16S539, D7S820).

While most PCR amplifications were

performed with quantitated genomic

DNA in liquid form, a few were tested with blood-stained FTATM paper

(Life Technologies, Rockville, MD).

Sample punches were removed from

the dried FTATM paper card with a 1.2

mm Harris MICRO-PUNCHTM (Life

Technologies). The recommended

washing protocol of 200 μL was

reduced to 25 or 50 μL in order to

reduce reagent costs and to work

with volumes that are compatible

with 96- or 384-well sample plates.

The number of washes was kept the

same as recommended by the manufacturer, but deionized water was

used instead of a 10 mM Tris-EDTA

solution. 

Two studies were performed with

larger numbers of DNA samples. In

collaboration with Dr. Steve Lee and

Dr. John Tonkyn from CDOJ’s DNA

research laboratory, CDOJ provided a

plate of 88 samples, which was used

repeatedly for multiple STR markers.

These anonymous samples had been

previously genotyped by CDOJ using

ABI’s AmpF1STR® ProfilerTM kit, which

consists of AmpF1STR® Blue, Green I,

and Yellow markers and amplifies 9

STRs and the sex-typing marker amelogenin. STR allelic ladders were also

provided by CDOJ and were used to

illustrate that the common alleles for

each STR locus could be detected with

GeneTrace primer sets. Researchers

retyped the samples using the

AmpF1STR® COfilerTM fluorescent STR

kit, which contains 6 STRs and amelogenin (5 of the 6 STR loci overlap 

with Profiler loci), thereby providing 

a further validation of each sample’s

true genotype. More recently, a set of

92 human DNA templates containing

3 different Centre d’Etude du Polymorphisme Humain (CEPH) families

(exhibit 25) and 44 unrelated individuals from the NIH Polymorphism

Discovery Resource were examined

(Collins et al., 1998). These samples

were typed on the ABI 310 Genetic

Analyzer using both the AmpF1STR®

Profiler PlusTM and AmpF1STR®

COfilerTM kits so that all 13 CODIS

STRs were covered.

PCR reaction

To speed the development of new STR

markers, researchers worked toward

the development of universal PCR 

conditions, in terms of both thermal

cycling parameters and reagents used.

Since almost all amplifications were

singleplex PCRs, development effort

was much simpler than multiplex PCR

development. Generally, all PCR reactions were performed in 20 μL volumes

with 20 pmol (1 μM) both forward and

reverse primers and a PCR reaction

mix containing everything else. The

CEPH Pedigree 104 (Venezuelan)

CEPH Pedigree 13293 (Utah)

CEPH Pedigree 884 (Amish)

Do not have

sample B3 C3 H2

A3

D3 B4 C4 D4 E4

A4

F4 E3 G4 H4 F3

B1

A1 A2

D2

D1 C2 E1 B2 F1 G1 H1

C1 E2

B5

D5 E5 F5 G6 H6 G5 H5 F2 G2

A6

C5 C6

D6 E6 F6

A5

B6

AM209

Exhibit 25. CEPH family pedigrees for samples examined. The 96 well 

positions are indicated for each sample. These samples were typed at 13 STRs 

and amelogenin using Profiler PlusTM and COfilerTM fluorescent STR kits. The 

samples were also typed at 8 STRs and amelogenin using the GeneTrace mass 

spectrometry primers. 
26

SCOPE AND METHODOLOGY

early PCR reaction mix contained 1 U

Taq polymerase (Promega); 1X STR

buffer with deoxynucleotide triphosphates (dNTPs) (Promega); and typically 5, 10, or 25 ng of human genomic

DNA. Later in the study, a PCR mix

containing 200 μM dNTPs, 50 mM

KCl, 10 mM Tris-HCl, 5% glycerol, and

2 mM MgCl2 was used. Typically, a

locus-specific master mix was prepared

by the addition of 12.8 μL of PCR mix

times the number of samples (+ ~10%

overfill) with 0.2 μL AmpliTaq GoldTM

DNA polymerase (ABI) and the appropriate volume and quantity of forward

and reverse primers to bring them to a

concentration of 1 μM in each reaction.

PCR reactions in a 96- or 384-well format were set up manually with an 8-

channel pipettor or robotically with a

Hamilton 16-tip robot.

Thermal cycling was performed in 96-

or 384-well MJ Research DNA Engine

(MJ Research, Watertown, MA) or 96

or dual block 384 PE9700 (ABI) thermal cyclers. 

Initial thermal cycling conditions

with Taq polymerase (Promega) were

as follows:

94 oC for 2 min

35 cycles:

94 oC for 30 sec

50, 55, or 60 oC for 30 sec

72 oC for 30 sec

72 oC for 5 or 15 min

4 oC hold

The final incubation at 72 oC favors

nontemplated nucleotide addition

(Clark 1988, Kimpton et al., 1993).

This final incubation temperature was

dropped to 60 oC for some experiments in an effort to drive the nontemplated addition even further. Later

experiments, including all of the larger

sample sets, involved using the following thermal cycling program with

TaqGold DNA polymerase:

95 oC for 11 min (to activate the 

TaqGold DNA polymerase)

40 cycles:

94 oC for 30 sec

55 oC for 30 sec

72 oC for 30 sec

60 oC for 15 min

4 oC hold

Primers were typically designed to have

an approximate annealing temperature

of 57–63 oC and thus worked well with

a 55 oC anneal step under this “universal” thermal cycling protocol. The need

for extensive optimization of primer

sets, reaction components, or cycling

parameters was greatly reduced or

eliminated with this approach for

primer development on STR markers.

Mitochondrial DNA samples were

amplified with 35 cycles and an annealing temperature of 60 o

C using the 

PCR primers listed in exhibit 26.

Multiplex PCR

Multiplex PCR was performed using 

a universal primer tagging approach

(Shuber et al., 1995; Ross et al.,

1998b) and the following cycling

program:

95 oC for 10 min

50 cycles:

94 oC for 30 sec

55 oC for 30 sec

68 oC for 60 sec

72 oC for 5 min

4 oC hold

The PCR master mix contained 5 mM

MgCl2, 2 U AmpliTaq Gold with 1X

Primer

Name

SNP Site

Position

SNP

Base* Sequence (5’ 3’)

Cleaved

 Mass (Da)

MT5 H16224 A/G b-GGAGTTGCAGTTGATGTGTGA(T)AGTTG 1,580

MT8’ L00146 T/C b-GTCGCAGTATCTGTCTTTGAT(T)CCTGCC 1,790

MT10 H00247 C/T b-CTGTGTGGAAAGTGGCTC(T)GCAGACATT 2,785

MT3’ H16189 A/G b-GGTTGATTGCTGTACTTGCTTG(T)AAGCATGGGG 3,179

MT9 H00152 A/G b-CTGTAATATTGAACGTAGG(T)GCGATAAATAAT 3,740

MT6 H16311 A/G b-GTGCTATGTACGG(T)AAATGGCTTTATGT 4,355

MT2g H16129 C/T b-GTACTACAGGTGG(T)CAAGTATTTATGGTAC 4,957

MT1 H16069 G/A b-AAATACA(T)AGCGGTTGTTGATGGGT 5,375

MT7 H00073 T/C b-CCAGCGTC(T)GCGAATGCTATCGCGTGCA 5,891

MT4e L00195 T/C b-CTACGT(T)CAATATTACAGGCGAACATAC 6,500

**PCR Primers produced a 1,021 bp PCR product spanning positions 15977–00422. 

DLOOP-F1** L15997 CACCATTAGCACCCAAAGCT

DLOOP-R1** H00401 CTGTTAAAAGTGCATACCGCCA

*Anderson reference sequence base listed first.

Exhibit 26. Mitochondrial DNA primers used for 10-plex SNP reaction

Note: “b” is listed for biotin; parentheses ( ) indicate the cleavable base.
27

SCOPE AND METHODOLOGY

Exhibit 27. Multiplex PCR primers used for Y SNP markers. Universal sequences have been attached to the 5’-end 

of the primers.

Primer

Name Primer Sequence (5’ 3’)

M2-F3u ATT TAG GTG ACA CTA TAG AAT ACG ACC CAG GAA GGT CCA GTA A

M2-R3u TAA TAC GAC TCA CTA TAG GGA GAC CCC CTT TAT CCT CCA CAG AT

M3-F1u ATT TAG GTG ACA CTA TAG AAT ACC TGC CAG GGC TTT CAA ATA G

M3-R1u TAA TAC GAC TCA CTA TAG GGA GAC TGA AAT TTA AGG GCA TCT TTC A

M13-F1u ATT TAG GTG ACA CTA TAG AAT ACT TAT GCC CAG GAA TGA ACA AG

M13-R1u TAA TAC GAC TCA CTA TAG GGA GAC CCA TGA TTT TAT CCA ACC ACA TT

M119-F1u ATT TAG GTG ACA CTA TAG AAT ACG GAA GTC ACG AAG TGC AAG T

M119-R1u TAA TAC GAC TCA CTA TAG GGA GAC GGG TTA TTC CAA TTC AGC ATA CAG

M35-F1u ATT TAG GTG ACA CTA TAG AAT ACA GGG CAT GGT CCC TTT CTA T

M35-R5u TAA TAC GAC TCA CTA TAG GGA GAC TGG GTT CAA GTT TCC CTG TC

M55-F1u ATT TAG GTG ACA CTA TAG AAT ACC AAA TAG GTG GGG CAA GAG A

M55-R1u TAA TAC GAC TCA CTA TAG GGA GAC CCT GGG ATT GCA TTT GTA CTT

M60-F1u ATT TAG GTG ACA CTA TAG AAT ACC CAA CAC TGA GCC CTG ATG

M60-R1u TAA TAC GAC TCA CTA TAG GGA GAC GAG AAG GTG GGT GGT CAA GA

M42-F1u ATT TAG GTG ACA CTA TAG AAT ACA GAT CAC CCA GAG ACA CAC AAA

M42-R1u TAA TAC GAC TCA CTA TAG GGA GAC GCA AGT TAA GTC ACC AGC TCT C

M67-F1u ATT TAG GTG ACA CTA TAG AAT ACG ACA AAC TCC CCT GCA CAC T

M67-R1u TAA TAC GAC TCA CTA TAG GGA GAC CTT GTT CGT GGA CCC CTC TA

M69-F1u ATT TAG GTG ACA CTA TAG AAT ACA CTC CTG GGT AGC CTG TTC A

M69-R1u TAA TAC GAC TCA CTA TAG GGA GAC GAA CCA GAG GCA AGG GAC TA

M26-F1u ATT TAG GTG ACA CTA TAG AAT ACC ACA GCA GAA GAG ACC AAG ACA

M26-R1u TAA TAC GAC TCA CTA TAG GGA GAC TGG GGC TGT ATT TGA CAT GA

M96-F1u ATT TAG GTG ACA CTA TAG AAT ACT GCC CTC TCA CAG AGC ACT T

M96-R1u TAA TAC GAC TCA CTA TAG GGA GAC AGA TTC ACC CAC CCA CTT TG

M122-F2u ATT TAG GTG ACA CTA TAG AAT ACA GTT GCC TTT TGG AAA TGA AT

M122-R2u TAA TAC GAC TCA CTA TAG GGA GAC GGT ATT CAG GCG ATG CTG AT

M145-F1u ATT TAG GTG ACA CTA TAG AAT ACG CTG GAG TCT GCA CAT TGA T

M145-R1u TAA TAC GAC TCA CTA TAG GGA GAC TGG ATC ATG GTT CTT GAT TAG G

M45-F2u ATT TAG GTG ACA CTA TAG AAT ACC ATC GGG GTG TGG ACT TTA C

M45-R2u TAA TAC GAC TCA CTA TAG GGA GAC ACA GTG GCA CCA AAG GTC AT

M9-F1u ATT TAG GTG ACA CTA TAG AAT ACA CTG CAA AGA AAC GGC CTA A

M9-R1u TAA TAC GAC TCA CTA TAG GGA GAC TTT TGA AGC TCG TGA AAC AGA

M89-F4u ATT TAG GTG ACA CTA TAG AAT ACC CAA ACA GCA AGG ATG ACA A

M89-R4u TAA TAC GAC TCA CTA TAG GGA GAC TGC AAC TCA GGC AAA GTG AG

M17-F1u ATT TAG GTG ACA CTA TAG AAT ACC TGG TCA TAA CAC TGG AAA TCA G

M17-R1u TAA TAC GAC TCA CTA TAG GGA GAC CCA CTT AAC AAA CCC CAA AAT

M130-F1u ATT TAG GTG ACA CTA TAG AAT ACG GGC AAT AAA CCT TGG ATT TC

M130-R1u TAA TAC GAC TCA CTA TAG GGA GAC GCA ATT TAG CCA CTG CTC TG

Univ-F: ATT TAG GTG ACA CTA TAG AAT AC

Univ-R: TAA TAC GAC TCA CTA TAG GGA GAC
28

SCOPE AND METHODOLOGY

M96 M45

M9

M89

M42

Remaining

primers

M130

M35

M96

M122

M45

M145

M9 M89

M42

5-plex

9-plex

(A)

Remove M35 and M122

Remove M130 and M35

Remove M145

Remove M35

Remove M130

Exhibit 28. CE electropherograms of “dropout” experiments conducted on a 9-plex PCR primer set used in 

developing Y-chromosome SNP markers. Section (A) demonstrates that by simply removing 4 primer pairs (M130, 

M35, M122, and M145), the multiplex PCR yield improves (i.e., there are fewer remaining primers and the amplicon 

yields are more balanced). Panel (B) shows that by removing only M130 and M35, the remaining primers are reduced to 

the greatest extent (see red oval). 

1.5

1.0

0.5

0.0

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5

5.0

2.5

0.0

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5

Minutes

4-02. Chan A

4-03. Chan A

4-05. Chan A

4-07. Chan A

4-10. Chan A

Remaining

primers

Minutes

(B)

Relative fluorescence units

Relative fluorescence units
29

SCOPE AND METHODOLOGY

PCR buffer II (ABI), 20 pmol of each

universal primer, and 0.2 pmol of each

locus-specific primer. The universal

primer sequences were 5’-ATTTAGGTGACACTATAGAATAC-3’ (attached 

on 5’ end of locus specific forward

primers) and 5’-TAATACGACTCACTATAGGGAGAC-3’ (attached on 5’

end of locus specific reverse primers).

Exhibit 27 shows the primer sequences

used for multiplex amplification of up

to 18 Y SNP markers. During multiplex PCR development studies, each

primer set was tested individually as

well as in the multiplex set. Primer

sets that were less efficient exhibited a

higher amount of remaining primers

or primer dimers in CE electropherograms of the PCR products. “Dropout” experiments, where one or more

primers were removed from the multiplex set, were then conducted to see

which primer sets interfered with one

another (exhibit 28). Finally, primer

concentrations were adjusted to try

and improve the multiplex PCR product balance between amplicons.

Verification of PCR 

amplification

Following PCR, a 1 μL aliquot of the

PCR product was typically checked on

a 2% agarose gel stained with ethidium

bromide to verify amplification success.

After a set of primers had been tested

multiple times and a level of confidence

had been gained for amplifying a particular STR locus, the gel PCR confirmation step was no longer used.

Later in this project, a Beckman

P/ACE 5500 capillary electrophoresis

(CE) instrument was used to check

samples after PCR. The quantitative

capabilities of CE are especially

important when optimizing a multiplex PCR reaction. As long as the

products are resolvable, their relative

peak area or heights can be used to

estimate amplification efficiency and

balance during the multiplex PCR

reaction. The CE separations were all

performed using an intercalating dye

and sieving polymer solution as previously described (Butler et al., 1995) to

avoid having to fluorescently label the

PCR products. Samples were prepared

for CE analysis by simply diluting a 1

μL aliquot of the amplicon in 49 μL of

deionized water.

SNP reaction and 

phosphatase treatment

For SNP samples, the amplicons were

treated with shrimp-alkaline phosphatase (SAP) (Amersham Pharmacia

Biotech, Inc., Piscataway, NJ) to

hydrolyze the unincorporated dNTPs

following PCR (Haff and Smirnov,

1997). Typically, 1 U of SAP was added

to each 20 μL PCR reaction and then

incubated at 37 o

C for 60 minutes 

followed by heating at 75 oC for 15

minutes. The SNP extension reaction

consisted of a 5 μL aliquot of the SAPtreated PCR product, 1X TaqFS buffer,

1.2–2.4 U TaqFS (ABI), 12.5 μM dideoxynucleotide triphosphate (ddNTP)

mix, and 0.5 μM biotinylated, cleavable

SNP primer in a 20 μL volume. For

multiplex analysis, SNP primer concentrations were balanced empirically, typically in the range of 0.3–1.5 μM, and

polymerase and ddNTP concentrations

were also doubled from the singleplex

conditions to facilitate extension from

multiple primers. The SNP extension

reaction was performed in a thermal

cycler with the following conditions: 

94 o

C for 1 min and 25–35 cycles at 94 o

C for 10 sec, 45–60 oC (depending on

the annealing temperature of the SNP

primer) for 10 sec, and 70 oC for 10 sec.

An annealing temperature of 52 oC was

used for the mtDNA 10plex SNP assay.

Sample Cleanup and

Mass Spectrometry

Following PCR amplification, a purification procedure involving solid-phase

capture and release from streptavidincoated magnetic beads was utilized

(Monforte et al., 1997) to remove 

salts that interfere with the MALDI ionization process (Shaler et al., 1996). At 

the start of this project, most of the

sample purification was performed

manually in 0.6 mL tubes with a 1.5

mL Dynal MPC®-E (Magnetic Particle

Concentrator for Microtubes of

Eppendorf Type) (Dynal A.S., Oslo,

Norway). Larger scale experiments

performed toward the end of this project utilized a robotic workstation fitted

with a 96-tip pipettor that mimicked

the manual method. This sample cleanup method involved washing the DNA

with a series of chemical solutions to

remove or reduce the high levels of

sodium, potassium, and magnesium

present from the PCR reaction. The

PCR products were then released from

the bead with a chemical cleavage step

that breaks the covalent bond between

the 5’-biotinylated portion of the DNA

product and the remainder of the extension product, which contains the STR

repeat region or the dideoxynucleotide

added during the SNP reaction. In the

final step prior to mass spectrometry

analysis, samples were evaporated to

dryness using a speed vac, reconstituted

in 0.5 μL of matrix (manual protocol)

or 2 μL of matrix (robotic protocol),

and spotted on the sample plate. 

The matrix typically used for STR

analysis was a 5:1 molar ratio of 

3-hydroxypicolinic acid (3-HPA)

(Lancaster Synthesis, Inc., Windham,

NH) with picolinic acid in 25 mM

ammonium citrate (Sigma-Aldrich, St.

Louis, MO) and 25% acetonitrile. For

SNP analysis, ~0.5 M saturated 3-HPA

was used with the same solvent of 25

mM ammonium citrate and 25% acetonitrile. A GeneTrace-designed and

built linear time-of-flight mass spectrometer was used as previously

described (Wu et al., 1994). Much

of the early data were collected manually on a research mass spectrometer.

During the time period of this project,

GeneTrace also built multiple highthroughput instruments.
30

SCOPE AND METHODOLOGY

Automated highthroughput mass 

spectrometer

GeneTrace has designed and custombuilt unique, automated time-of-flight

mass spectrometers for high-throughput

DNA analysis. The basic instrument

design is covered under U.S. Patent

5,864,137 (Becker and Young, 1999).

A high repetition rate UV laser (e.g.,

100 Hz) is used to enable collection of

high quality mass spectra consisting of

100–200 summed shots in only a few

seconds. The sample chamber can

hold up to two sample plates at a time

with each plate containing 384 spotted

samples. Exhibit 29 shows a sample

plate on the X–Y table under the custom GeneTrace ion optics. 

An important feature of this automated

mass spectrometer is “peak picking”

software that enables the user to define

“good” versus “bad” mass spectra. After

each laser pulse, the “peak picker”

algorithm checks for peaks above a

user-defined signal-to-noise threshold

in a user-defined mass range. Only

“good” spectra are kept and summed

into the final sample spectrum, which

improves the overall signal quality.

The X–Y table moves in a circular pattern around each sample spot until

either the maximum number of good

shots (e.g., 200) or the maximum

number of total shots (e.g., 1,000) is

reached. A mass spectrum’s signal-tonoise level is related to the number of

laser shots collected. In general, signalto-noise improves as the square root

of the number of shots. Thus, improving the signal by a factor of two would

require increasing the number of good

shots collected by a factor of four.

Raw data files (.dat) were converted 

to “smoothed” data files (.sat) using

custom software developed at GeneTrace that improved data quality and

involved several multipoint SavitzkyGolay averages along with a baseline

subtraction algorithm (Carroll and

Beavis, 1996). A set of samples was

collected under a single “header” file

with identical peak picking parameters.

Each header file recorded the mass

calibration constants and peak picking

parameters and listed all of the samples

analyzed with the number of good

shots collected versus the number of

total shots taken for each sample. 

Data points in mass spectrometry are

collected in spectral channels that

must be converted from a time value

to a mass value. This mass calibration

is normally performed with two

oligonucleotides that span the mass

range being examined. For example,

a 36-mer (10,998 Da) and a 55-mer

Exhibit 29. Photo of the automated GeneTrace mass spectrometry ion 

optics over a sample plate containing 384 different DNA samples. A 

sample in the center of the plate is illuminated by a pulsing laser light.
31

SCOPE AND METHODOLOGY

(16,911 Da) were typically used when

examining STRs in the size range of

10,000–40,000 Da. On the other hand,

a 15-mer (4,507 Da) and its doubly

charged ion (2,253.5 Da) were used

to cover SNPs in the size range of

1,500–7,500 Da. Ideally, larger mass

oligonucleotides would be used for

STR analysis to obtain more accurate

masses, but producing a clean, wellresolved peak above 25–30 kDa is a

synthetic and instrumental challenge.

The calibration was typically performed

only once per day because the calibration remains consistent over hundreds

of samples. The mass accuracy and

precision are such that no sizing standards or allelic ladders need to be run

to determine a sample’s size or genotype (Butler et al., 1998b).

Delayed extraction (Vestal et al., 1995)

and mass gating (“blanking”) were

used to improve peak resolution and

sensitivity, respectively. Typically, a

delay of 500–1,000 nanoseconds was

used to eliminate ions below ~8,000

Da for STRs, and a delay of 250–500

nanoseconds was used with a signal

blanking below ~1,000 Da for SNPs.

Sample Genotyping

Automated STR 

genotyping program

(CallSSR or CallSTR)

During the time period of this project,

GeneTrace developed an automated

sample genotyping program, named

CallSSR. The data sets described in

the Results section were processed

either with CallSSR version 1.82 or

a modified version of the program

named CallSTR. The program was

written in C++ at GeneTrace by a scientific programmer named Nathan

Hunt and can run on a Windows® NT

platform. A reference DNA sequence

is used to establish the possible STR

alleles and their expected masses based

on an expected repeat mass and range

of alleles. This mass information is

recorded in a mass ladder file (exhibit

30). In the case of the forensic STR

loci examined in this project, the

GenBank sequences were used as the

reference DNA sequences. 

Locus Repeat Reference Reference Minimum Maximum Forward Reverse

Name Mass (Da) Allele Mass (Da) Repeat Repeat Primer Primer

TH01 1,260 9 21,133 3 12

12

12

12

14

14

16

16

16

16

16

21

21

21

16

16

16

16

16

30

18

14

7

7

TH01-F2 TH01-R2-GTS

TH01b 1,260 9 18,685 3 TH01-F TH01-R-GTS

TH01b2 1,260 9 18,372 3 TH01-F (w/o+A) TH01-R-GTS

TH01c 1,260 9 19,856 3 ddC TH01-R2-GTS

TPOX 1,260 11 21,351 5 TPOX-F-GTS TPOX-R

TPOXc 1,260 11 15,119 5 TPOX-F-GTS ddC

CSF 1,260 12 28,890 5 CSF-F3-GTS CSF-R3

CSFb 1,211 12 27,989 5 CSF-F3 CSF-R3-GTS

CSFc 1,260 12 22,993 5 CSF-F3-GTS ddC

AMEL 951 6 27,333 3 AMEL-F-GTS AMEL-R

AMELb 924 6 18,239 4 AMEL-F2 AMEL-R2-GTS

D7S820 1,260 12 21,639 5 D7S820-F-GTS D7-R

D7S820c 1,260 12 16,949 5 D7S820-F-GTS ddC

D3S1358 1,211 16 34,300 9 D3-F D3-R-GTS

D3S1358b 1,260 15 25,624 9 D3-F2-GTS D3-R2

D3S1358c 1,260 15 15,994 9 D3-F2-GTS ddC

D16S539 1,211 11 18,180 4 D16-F D16-R-GTS

D16S539b 1,260 11 26,668 4 D16-F4-GTS D16-R4

D16S539c 1,260 11 14,765 4 D16-F4-GTS ddC

D16S539d 1,211 11 25,572 4 D16-F4 D16-R4-GTS

D16S539e 1,211 11 19,127 4 D16-F D16-R2-GTS

FGA 1,202 21 37,180 15 FGA-F2-GTS FGA-R2

D8S1179 1,211 12 24,417 8 D8S1179-F-GTS D8S1179-R

DYS391 1,211 9 23,004 7 DYS391-F-GTS DYS391-R

Exhibit 30. Mass ladder file for STR loci analyzed in this study. Different masses exist for a locus due to different 

primer positions. The most commonly used primer sets are highlighted. The reference mass does not include nontemplate 

addition by the polymerase. The genotyping program automatically adds 313 Da to the reference mass for compensation of 

adenylation. 
32

SCOPE AND METHODOLOGY

CallSSR accepts as input smoothed,

baseline-subtracted data files, a “layout”

file, and the mass ladder file. The layout file describes each sample’s position on the 384-well plate, the primer

set used for PCR (i.e., the STR locus),

and the DNA template name. The program processes samples at a rate of

more than one sample per second so

that a plate of 384 samples can be

genotyped in less than 5 minutes.

This high rate of processing speed is

necessary in a high-throughput 

environment where thousands of samples must be genotyped every day.

Two files result from running the program: a “call file” and a “plot file.”

The call file may be imported into

Microsoft® Excel for data examination

and contains information like the

allele mass and calculated sample

genotype. The plot file generates 

plotting parameters that work with

MATLAB (The Math Works, Inc.,

Natick, MA) scripts to plot 8 mass

spectra per page, as seen in exhibit 31.

Plots are generated in an artificial

repeat space to aid visual inspection 

of the mass spectrometry data compared with allele bins. The CallSSR

algorithm has been written to ignore

stutter peaks and double-charged

peaks, which are artifacts of the DNA

amplification step and mass spectrometry ionization process, respectively. 

Exhibit 31. Mass spectra of CDOJ samples amplified with CSF1PO primers. From left side, top-to-bottom 

followed by right side, top-to-bottom, sample genotypes are (7,11), (8,10), (10,10), (9,10), (12,12), (11,12), (12,14), 

and allelic ladder. The mass range shown here is 15,000–35,000 Da.

1.5

 x 104

 x 104

 x 104

Mass (Da)

Intensity (arbitrary units)

Mass (Da)

Intensity (arbitrary units)

Mass (Da)

Mass (Da)

Intensity (arbitrary units)

Intensity (arbitrary units)

Intensity (arbitrary units) Intensity (arbitrary units)

Intensity (arbitrary units)

Intensity (arbitrary units)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

2 2.5 3 3.5 1.5 2 2.5 3 3.5

1.5 2 2.5 3 3.5 1.5 2 2.5 3 3.5

1.5 2 2.5 3 3.5 1.5 2 2.5 3 3.5

1.5 2 2.5 3 3.5 1.5 2 2.5 3 3.5

1

 x 104

 x 104

 x 104

 x 104

1

 x 104

0

0

1

2

0

1

2

0

1

2

0

0

2

0

1

2

2

0

 x 104 x 104

4

2

2 4
33

SCOPE AND METHODOLOGY

Automated SNP 

genotyping program

(CallSNP)

In-house automated SNP analysis 

software was developed and used to

determine the genotype for each SNP

marker. This program, dubbed CallSNP,

was written in C++ by a scientific programmer named Kevin Coopman and

will run on a Windows NT or UNIX

platform. The software searches for an

expected primer mass and, after locating the pertinent primer, searches for

the four possible extension products by

using a linear least squares fit, with the

primer peak shape serving as the fitting

line. In this way, peak adducts from the

ionization process are distinguished

from true heterozygotes. The fit coefficients of the four possible nucleotides

are then compared with one another to

determine the appropriate SNP base.

The base with the highest value (i.e.,

best fit) is the called base. The mass

between the primer and the extension

product can then be correlated to the

incorporated nucleotide at the SNP

site. In the case of a heterozygote at the

SNP site, two extension products exist

and are called by the software. 

As with the CallSSR software, a layout

file, a mass file, and mass spectrometry

data files are required as input. Call

files generate information regarding the

closeness of the fit for each possible

nucleotide with an error value associated for each call. The SNP mass information file includes the SNP marker

name, expected primer mass (postcleavage), and expected SNP bases.

The current version of CallSNP works

well for singleplex SNPs but needs

modifications before it can work effectively on multiplex SNP samples. In

principle, the program could be scaled

to limited, widely spaced multiplexes

where the doubly charged ions of larger mass peaks do not fall in the range

of lower mass primer peaks.

Comparison Tests 

With ABI 310 Genetic

Analyzer

For comparison purposes, more than

200 genomic DNA samples were genotyped using the Applied Biosystems

310 Genetic Analyzer and the

AmpF1STR® Profiler PlusTM or

AmpF1STR® COfilerTM fluorescent

STR kit. Exhibit 32 lists the numbers

of samples analyzed with each STR

kit. STR samples were run in the ABI

310 CE system using the POP-4 polymer, 1X Genetic Analysis buffer, and

Sample Set

Number 

of Samples Kit Used MS Data Compared Comments

DOJ plate 88 COfiler TH01, TPOX, CSF1PO,

D7S820, D3S1358, D16S539, 

D8S1179, FGA, DYS391, and 

amelogenin 

See exhibits 12–19, 39,

and 40

CEPH/Diversity

plate

92 COfiler and

ProfilerPlus

TH01, TPOX, CSF1PO,

D7S820, D3S1358, D16S539,

D8S1179, and amelogenin 

See exhibit 63

Stanford male

samples

37 ProfilerPlus For Y SNP testing

(not completed)

See exhibits 44–45; new 

microvariants in D18, 

D21, FGA seen 

Butler family samples 34 COfiler

JMB family samples 4 COfiler and 

ProfilerPlus

Standard templates 3 COfiler and

ProfilerPlus

TH01, TPOX, CSF1PO, 

D7S820, D3S1358, D16S539, 

D8S1179, FGA, DYS391, 

and amelogenin 

See exhibit 73

K562, AM209, and UP006

TOTAL TESTED 258 221 COfiler

136 ProfilerPlus 2,907 total genotypes measured

CDOJ results 88 Profiler (under 

CDOJ validated 

method) 

See above B11, D3S1358:15,15.2

incorrectly called 14,15

(see exhibit 74)

Exhibit 32. Sample sets run on ABI 310 Genetic Analyzer with AmpF1STR®

 Profiler Plus or AmpF1STR®

COfiler fluorescent STR kits 

No mass spectrometry data collected

No mass spectrometry data collected

TM 

TM 
34

SCOPE AND METHODOLOGY

a 47-cm (50 μm i.d.) capillary with

the GS STR POP4 (1 mL) F separation module (ABI Prism 310 Genetic

Analyzer User’s Manual, 1998). With

this module, samples were electrokinetically injected for 5 seconds at

15,000 volts and separated at 15,000

volts for 24 minutes with a run temperature of 60 o

C. DNA sizing was

performed with ROX-labeled GS500

as the internal sizing standard. Samples

were prepared by adding 1 μL PCR

product to 20 μL deionized formamide

containing the ROX-GS500 standard.

The samples were heat-denatured at

95 oC for 3 minutes and then snap

cooled on ice prior to being loaded

into the autosampler tray. These separation conditions and sizing standards

are commonly used in validated protocols by forensic DNA laboratories.

Following data collection, samples

were analyzed with Genescan 2.1 and

Genotyper 2.0 software programs (ABI).

While standard CE conditions were

used, new PCR conditions were 

developed to dramatically reduce the

cost of using ABI’s STR kits. The PCR

volume was reduced from the standard

50 μL described in the ABI protocol

(AmpF1STR® ProfilerPlusTM, 1998, and

AmpF1STR® CofilerTM, 1998) to 5 μL,

which corresponded with a cost reduction of 90% per DNA amplification.

The kit reagents were mixed in their

ABI-specified proportions—11 μL

primer mix, 1 μL TaqGold polymerase

(5 U/μL), and 21 μL PCR mix. A 3 μL

aliquot of this master mix was then

added to each tube along with 2 μL of

genomic DNA template (typically at

1–2 ng/μL). Both PE9700 and MJ

Research thermal cyclers worked for

this reduced PCR volume method 

provided that the 200 μL PCR tubes

were sealed well to prevent evaporation. Results showed that an 8-strip

of 0.2 mL thin wall PCR tubes from

Out Patient Services, Inc., (OPS)

(Petaluma, CA) worked best for the

PE9700 thermal cycler. Only 1 μL is

needed for CE sample preparation; a

sample that can be re-injected multiple

times if needed. Thus, with a 50 μL

PCR reaction, 49 μL were never used

to produce a result under the standard

ABI protocol. A 5 μL PCR produces

less waste in addition to being less

expensive. More importantly, the 

multiplex STR amplicons were more

concentrated in a lower volume and

produced higher signals in the ABI

310 data collection. Peak signals were

often off-scale, and the number of

cycles in the cycling program could

be reduced from 28 to 26, or even 

25 with some DNA templates. This

5 μL PCR also worked well with FTA

paper punches that have been washed

with FTA purification reagent (Life

Technologies).

The California Department of Justice

ran one plate of 88 samples with the

AmpF1STR® ProfilerTM kit on an ABI

310 Genetic Analyzer and provided

those genotypes for comparison purposes. These results provided an independent verification of our work.
35

RESULTS AND DISCUSSION OF STR ANALYSIS

BY MASS SPECTROMETRY

I

n the course of this work, thousands of data points were collected

using STR markers of forensic

interest verifying that GeneTrace’s

mass spectrometry technology works.

During this same time, tens of thousands of data points were gathered

across hundreds of different microsatellite markers from corn and soybean

as part of an ongoing plant genomics

partnership with Monsanto Company

(St. Louis, MO). Whether the DNA

markers used come from humans or

plants, the characteristics described

below apply when analyzing polymorphic repeat loci.

Marker Selection and

Feasibility Studies

With STR Loci

Prior to receiving grant funding, feasibility work had been completed using

the STR markers TH01, CSF1PO,

FES/FPS, and F13A1 in the summer

and fall of 1996 (Becker et al., 1997).

At the start of this project, a number

of STR loci were considered as possible candidates to expand upon the

initial four STR markers and to develop a set of markers that would work

well in the mass spectrometer and

would be acceptable to the forensic

DNA community. Searches were made

of publicly available databases, including the Cooperative Human Linkage

Center (http://lpg.nci.nih.gov/CHLC),

the Genome Database (http://gdbwww.

gdb.org), and Weber set 8 of the

Marshfield Medical Research Foundation’s Center for Medical Genetics

(http://www.marshmed.org/genetics).

Literature was also searched for possible tetranucleotide markers with

PCR product sizes below 140 bp in

size to avoid having to redesign the

PCR primers to meet our limited size

range needs (Hammond et al., 1994;

Lindqvist et al., 1996). The desired

characteristics also included high heterozygosity, moderate number of alleles (<7 or 8 to maintain a narrow mass

range) with no known microvariants

(to avoid the need for a high degree

of resolution), and balanced allele frequencies (most commonly allele <40%

and least common allele >5%). This

type of marker screen was found to be

rather inefficient because the original

primer sets reported in public STR

databases were designed for gel-based

separations, which were optimal over a

size range of 100–400 bp. In fact, most

of the PCR product sizes were in the

200–300 bp range. From a set of several thousand publicly available STRs,

only a set of eight candidate tetranucleotide STRs were initially identified;

three of which were tested using the

original reported primers (exhibit 33).

The initial goal was to identify ~25

markers that spanned all 22 autosomal

chromosomes as well as the X and Y

sex chromosomes. 

Researchers quickly realized that population data were not available on

these “new” markers and would not 

be readily accepted without extensive

testing and validation. Since one of

the objectives was to produce STR

marker sets that would be of value to

the forensic DNA community, the next

step taken was the examination of STR

markers already in use. After selecting

Marker Name Heterozygosity Size Range

Allele

Frequencies

Number of

Alleles

GenBank

Sequence

D1S1612 0.83 94–134 bp 0.9–26% 10 alleles G07863

D2S1391 0.79 109–137 bp 5–35% 8 alleles G08168

D5S1457 0.74 97–127 bp 1–32% 8 alleles G08431

GATA132B04

(D5S2843)

Not reported 98–114 bp 12–35% 5 alleles G10407

D16S2622 Not reported 71–91 bp 3–54% 5 alleles G07934

D16S764 0.70 96–116 bp 4–38% 5 alleles G07928

D19S591 0.74 96–112 bp 6–36% 5 alleles G09745

D22S445 0.65 110–130 bp 3–37% 6 alleles G08096

Exhibit 33. Tetranucleotide markers identified through literature and public database searches as possible candidates 

for early STR marker development. The databases searched included the Cooperative Human Linkage Center, Marshfield 

Clinic Weber set 8, and the Genome Database. Primers were synthesized and tested for the markers in bold. 
36

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

STR markers used by the Promega

Corporation, Applied Biosystems, and

the Forensic Science Service (FSS),

researchers redesigned primer pairs

for each STR locus to produce smaller

PCR product sizes. These STR markers included TPOX, D5S818, D7S820,

D13S317, D16S539, LPL, F13B, HPRTB,

D3S1358, VWA, FGA, CD4, D8S1179,

D18S51, and D21S11. The primers

for TH01 and CSF1PO were also

redesigned to improve PCR efficiencies

and to reduce the amplicon sizes.

Primers for amelogenin, a commonly

used sex-typing marker, were also tested (Sullivan et al., 1993). In addition,

two Y-chromosome STRs, DYS19 and

DYS391, were examined briefly. Exhibit

34 summarizes the STR primer sets

that were developed and tested over

the course of this project. However,

with the announcement of the 13

CODIS core loci in the fall of 1997,

emphasis switched to CSF1PO, TPOX,

TH01, D3S1358, VWA, FGA, D5S818,

D7S820, D13S317, D16S539,

D8S1179, D18S51, D21S11, and the

sex-typing marker amelogenin.

The newly designed GeneTrace

primers produced smaller PCR products than those commercially available

from Applied Biosystems or Promega

(exhibit 2), yet resulted in identical

genotypes in almost all samples tested.

For example, correct genotypes were

obtained on the human cell line K562,

a commonly used control for PCR

amplification success. Exhibit 35

shows the K562 results for CSF1PO,

TPOX, TH01, and amelogenin. These

results were included as part of a 

publication demonstrating that timeof-flight mass spectrometry could perform accurate genotyping of STRs

without allelic ladders (Butler et al.,

1998).

Caveats of STR analysis 

by mass spectrometry

While mass spectrometry worked well

for a majority of the STR markers tested, a few limitations excluded some

STRs from working effectively. Two

important issues that impact mass

spectrometry results are DNA size

and sample salts. Mass spectrometry

resolution and sensitivity are diminished when either the DNA size or

the salt content of the sample is too

large (Ross and Belgrader, 1997; and

Taranenko et al., 1998). By designing

the PCR primers to bind close to the

repeat region, the STR allele sizes are

reduced so that resolution and sensitivity of the PCR products are benefited. In addition, the GeneTrace-patented

cleavage step reduces the measured

DNA size even further. When possible,

primers are designed to produce amplicons that are less than 120 bp, although

work is sometimes undertaken with

STR alleles that are as large as 140 bp

in size. This limitation in size prevents

reliable analysis of STR markers with

samples containing a large number

of repeats, such as most of the FGA,

D21S11, and D18S51 alleles (exhibit 2). 

To overcome the sample salt problem,

researchers used a patented solid-phase

purification procedure that reduced the

concentration of magnesium, potassium, and sodium salts in the PCR products prior to being introduced to the

mass spectrometer (Monforte et al.,

1997). Without the reduction of the

salts, resolution is diminished by the

presence of adducts. Salt molecules

bind to the DNA during the MALDI

ionization process and give rise to

peaks that have a mass of the DNA

molecule plus the salt molecule.

Adducts broaden peaks and thus

reduce peak resolution. The sample

Human

Chromosome STR Marker

Human

Chromosome STR Marker

1 F13B 13 D13S317

2 TPOX 14

3 D3S1358 15 FES/FPS

4 FGA 16 D16S539, D16S2622

5 CSF1PO, D5S818,

GATA132B04

17

6 F13A1 18 D18S51

7 D7S820 19

8 LPL, D8S1179 20

D21S11

10 22 D22S445

11 TH01 X HPRTB, Amelogenin

12 VWA, CD4 Y DYS19, DYS391,

Amelogenin

Exhibit 34. STR markers examined at GeneTrace during the course of this 

project as sorted by their chromosomal position. Primers were designed, 

synthesized, and tested for each of these markers. The most extensive testing 

was performed with the markers highlighted in green. Amelogenin, which is 

a gender identification marker rather than an STR, is listed twice because it 

occurs on both the X and Y chromosomes. The italicized STRs are those not 

commonly used by the forensic DNA community. 

9 21
37

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

purification procedure, which was

entirely automated on a 96-tip robotic

workstation, reduced the PCR buffer

salts and yielded “clean” DNA for the

mass spectrometer. Appropriate care

must be taken to prevent samples from

being contaminated with salts both

during and after the sample purification procedure.

Size reduction methods

The portion of the DNA product on

the other side of the repeat region from

the cleavable primer was removed in

one of two possible ways: using a

restriction enzyme (Monforte et al.,

1999) or performing a nested linear

amplification with a ddN terminating

nucleotide (Braun et al., 1997a and

1997b). Both methods have pros and

cons. A restriction enzyme, DpnII,

which recognizes the sequence 5’...

^GATC...3’, was used with VWA samples to remove 45 bp from each PCR

product. For example, the GenBank

allele that contains 18 repeat units and

is 154 bp following PCR amplification

may be reduced to 126 nucleotides following primer cleavage, but it can be

shortened to 81 nucleotides if primer

cleavage is combined with DpnII digestion. At 81 nt or 25,482 Da, the STR

product size is much more manageable in the mass spectrometer. This

approach works nicely provided the

restriction enzyme recognition site

remains unchanged. The DpnII digestion of VWA amplicons worked on all

samples tested, including a reamplification of an allelic ladder from ABI

(exhibit 36). However, the cost and

time of analysis are increased with the

addition of a restriction enzyme step.

The second approach for reducing the

overall size of the DNA molecule in

the mass spectrometer involved using

a single ddNTP with three regular

dNTPs. A linear amplification extension reaction was performed with the

ddNTP terminating the reaction on

the opposite side of the repeat from

the cleavable primer. However, there

Exhibit 35. Mass spectra for CSF1PO, TPOX, TH01, and amelogenin 

using K562 DNA. Genotypes agree with results reported by the manufacturer 

(Promega Corporation). The numbers above the peaks represent the allele 

calls based upon the observed mass. The allele imbalance on the heterozygous 

samples is because the K562 strain is known to contain an unusual number of 

chromosomes and some of them are represented more than twice per cell. The 

TH01 peak is split because it is not fully adenylated (Butler et al., 1998). 

15,000 20,000 25,000 30,000

CSF1PO

TPOX 9

8

9

10

9.3 TH01

Signal

Amelogenin X

Mass (Da)
38

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

were several limitations with this “single base sequencing” approach. First,

it only worked if the repeat did not

contain all four nucleotides. For example, a nucleotide mixture of dideoxycytosine (ddC), deoxyadenosine (dA),

deoxythymidine (dT), and deoxyguanosine (dG) will allow extension

through an AATG repeat (as occurs in

the bottom strand of TH01) but will

terminate at the first C nucleotide in a

TCAT repeat (the top strand of TH01).

Thus one is limited with the DNA

strand that can be used for a given

combination of dideoxynucleotide and

corresponding deoxynucleotides. In

addition, primer position and STR

sequence content are important. If a

ddC mix is used, the DNA sample cannot contain any C nucleotides prior to

the repeat region or within the repeat,

or the extension will prematurely halt

and the information content of the full

repeat will not be accurately captured.

In most cases, this requires the extension primer to be immediately adjacent to the STR repeat, a situation that

is not universally available due to the

flanking sequences around the repeat

region. For example, this approach

will work with TH01 (AATG) but not

VWA, which has three different repeat

structures: AGAT, AGAC, and AGGT.

Thus with VWA, a ddC would extend

through the AGAT repeat but would

be prematurely terminated at the C in

the AGAC repeat, and valuable polymorphic information would be lost.

The use of a terminating nucleotide

also provides a sharper peak for an

amplified allele compared with the split

peaks or wider peaks (if resolution is

poor) that can result from partially

adenylated amplicons (i.e., -A/+A).

Exhibit 37 illustrates the advantage of

a ddG termination on a D8S1179 heterozygous sample containing 11 and

13 TATC repeats. In the bottom panel,

23 nt were removed compared with

the top panel, which corresponds to

a mass reduction of almost 8,000 Da.

The peaks are sharper in the lower

panel, as the products are blunt ended.

Identical genotypes were obtained

with both approaches, illustrating that

the ddG termination is occurring at

the same point on the two different

sized alleles.

To summarize, STR sample sizes were

reduced using primers that have been

designed to bind close to the repeat

region or even partially on the repeat

itself. A cleavable primer was incorporated into the PCR product to allow

post-PCR chemical cleavage and 

subsequent mass reduction. Two 

additional post-PCR methods were

also explored to further reduce the

measured DNA size. These methods

20,000 22,000 24,000 26,000 28,000 30,000 32,000

14,000 16,000 18,000 20,000 22,000 24,000

14,000 16,000 18,000 20,000

16,000 18,000 20,000 22,000 24,000 26,000 28,000 30,000

Exhibit 36. Mass spectra of STR allelic ladders from CSF1PO, TPOX, 

TH01, and VWA. The numbers above each peak designate the allele name 

(number of repeats). Peak widths vary among samples based on DNA size 

and salt content. Smaller sizes (e.g., TH01) give sharper peaks than larger 

sizes (e.g., CSF1PO). On a mass scale as shown here, each nucleotide is 

approximately 300 Daltons (Da). The VWA ladder was digested with DpnII 

restriction enzyme following PCR to reduce the overall size of the amplicons. 

CSF1PO

TPOX

TH01

VWA

6

7

8 9

10

11

12

13

15

14

6 7 8 9

10 11 12

13

6 7

8 9 10

5 9.3

11 12 15

16 17 18

13

14

19 20 21

Mass (Da)
39

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

included restriction enzyme digestion

in the flanking region on the other

side of the repeat region from the

cleavable primer and a primer extension through the repeat region with a

single dideoxynucleotide terminator

(single base sequencing approach).

To illustrate the advantages of these

approaches to reduce the overall

DNA product mass, researchers examined the STR locus TPOX. Using a

conventional primer set, a sample

containing 11 repeats measured 232

bp or ~66,000 Da. By redesigning the

primers to anneal close to the repeat

region, a PCR product of 89 bp was

obtained. With the cleavable primer,

the size was reduced to 69 nt or

21,351 Da. By incorporating a ddC

termination reaction, another 20 nt

were removed leaving only 49 nt or

~12,000 Da (primarily only the repeat

region). The repeat region contained

44 nt (4 nt x 11 repeats) or ~10,500

Da. The ddC termination was also

used in multiplex STR analysis to

produce a CSF1PO-TPOX-TH01

triplex (exhibits 4 and 5). The repeat

sequences used for these STR loci were

AGAT for CSF1PO, AATG for TPOX,

and AATG for TH01. The level of

sequence clipping by ddC was as 

follows: CSF1PO (-14 nt), TPOX 

(-20 nt), and TH01 (-4 nt). 

-A/+A

Mass reduced by 7,477 Da

Exhibit 37. Mass spectra of a D8S1179 sample illustrating the benefit of a dideoxynucleotide termination approach. 

The top panel displays a result from a regular PCR product; the bottom panel contains the same sample treated with a linear 

amplification mix containing a ddG terminator with dA, dT, and dC deoxynucleotides. With the ddG approach, the problem 

of incomplete adenylation (both –A and +A forms of a PCR product) is eliminated, and the amplicons are smaller, which 

improves their sensitivity and resolution in the mass spectrometer. The red oval highlights the broader -A/+A peaks present 

in the regular PCR product. Note: The genotype (i.e., 11 and 13 repeats) is identical between the two approaches, even 

though almost 8,000 Da are removed with the ddG termination. 

12,000 14,000 16,000 18,000 20,000 22,000 24,000 26,000 28,000 30,000

11

13

(not fully adenylated)

Signal

Mass (Da)

11

13

ddG approach

regular PCR products
40

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

Multiplex STR Work

Due to the limited size range of DNA

molecules that may be analyzed by

this technique, a new approach to

multiplexing was developed that

involved interleaving alleles from

different loci rather than producing

nonoverlapping multiplexes. If the

amplicons could be kept under

~25,000 Da, a high degree of mass

accuracy and resolution could be used

to distinguish alleles from multiple

loci that may differ by only a fraction

of a single nucleotide (exhibit 10).

Allelic ladders are useful to demonstrate that all alleles in a multiplex

are distinguishable (exhibit 9).

The expected masses for a triplex

involving the STR loci CSF1PO,

TPOX, and TH01 (commonly referred

to as a CTT multiplex) are schematically displayed in exhibit 4. All known

alleles for these STR loci, as defined by

STRBase (Ruitberg et al., 2001), are

fully resolvable and far enough apart

to be accurately determined. For

example, TH01 alleles 9.3 and 10 fall

between CSF1PO alleles 10 and 11.

For all three STR systems in this CTT

multiplex, the AATG repeat strand is

measured, which means that the alleles within the same STR system differ

by 1,260 Da. The smallest spread

between alleles across multiple STR

systems in this particular multiplex

exists between the TPOX and TH01

alleles, where the expected mass difference is 285 Da. TPOX and CSF1PO

alleles differ by 314 Da, while TH01

and CSF1PO alleles differ by 599 Da.

By using the same repeat strand in the

multiplex, the allele masses between

STR systems all stay the same distance

apart. Each STR has a unique flanking

region and it is these sequence differences between STR systems that permit multiplexing in such a fashion as

described here. An actual result with

this CTT multiplex is shown in exhibit 5. This particular sample is homozygous for both TPOX (8,8) and CSF1PO

(12,12) and heterozygous at the TH01

locus (6,9.3).

It is also worth noting that this particular CTT multiplex was designed to

account for possible, unexpected

microvariants. For example, a CSF1PO

allele 10.3 that appears to be a single

base shorter than CSF1PO allele 11

was recently reported (Lazaruk et al.,

1998). With the CTT multiplex primer

set described here, a CSF1PO 10.3

allele would have an expected mass of

21,402 Da, which would be fully distinguishable from the nearest possible

allele (i.e., TH01 allele 10) because

these alleles would be 286 Da apart.

Using a mass window of 100 Da as

defined by previous precision studies

(Butler et al., 1998), all possible alleles

including microvariants should be

fully distinguishable. STR multiplexes

are designed so that expected allele

masses between STR systems are offset

in a manner that possible microvariants, which are most commonly insertions or deletions of a partial repeat

unit, may be distinguished from all

other possible alleles. The larger the

allele’s mass range, the more difficult it

becomes to maintain a high degree of

mass accuracy. For example, exhibit 8

shows the observed mass for TH01

allele 9.3 is -52 Da from its expected

mass, while TPOX allele 9 is only 3

Da from its expected mass. In this particular case, the mass calibrants used

were 4,507 Da and 10,998 Da. Thus,

the TPOX allele’s mass measurement

was more accurate and closer to the

calibration standard. The ability to

design multiplexes that have a relatively compact mass range is important

to maintaining the high level of mass

accuracy needed for closely spaced

alleles from different, overlapping STR

loci. The mass calibration standards

should also span the entire region of

expected measurement to guarantee

the highest degree of mass accuracy.

Two possible multiplexing strategies

for STR genotyping are illustrated

in exhibit 38. Starting with a single

punch of blood stained FTA paper, it

is possible to perform a multiplex PCR

(simultaneously amplifying all STRs 

of interest) followed by another PCR

with primer sets that are closer to the

repeat region. With this approach,

single or multiplexed STR products

can be produced that are small

enough for mass spectrometry analysis. Alternatively, multiple punches

could be made from a single bloodstain on the FTA paper followed by

singleplex or multiplex PCR with mass

spectrometry primers. After the genotype is determined for each STR locus

in a sample, the information would be

combined to form a single sample

genotype for inclusion in CODIS or

some other DNA database. This multiplexing approach permits flexibility

for adding new STR loci or only processing a few STR markers across a

large number of samples at a lower

cost than processing extensive and

inflexible STR multiplexes.

Comparison Tests

Between ABI 310 and

Mass Spectrometry

Results

A plate of 88 samples from the CDOJ

DNA Laboratory was tested with 10

different STR markers and compared

with results obtained using the ABI

310 Genetic Analyzer and commercially available STR kits. The samples

were supplied as a 200 μL aliquot of

extracted genomic DNA in a 96-well

tray with each sample at a concentration of 1 ng/μL. A 5 μL aliquot was

used for each PCR reaction, or 5 ng

total per reaction. Since each marker

was amplified and examined individually, approximately 35 ng of extracted

genomic DNA was required to obtain

genotypes on the same 7 markers as

were amplified in a single AmpF1STR®

COfilerTM STR multiplex. Only 2 ng of

genomic DNA were used per reaction

with the AmpF1STR® COfilerTM kit.

Thus, a multiplex PCR reaction is much

better suited for situations where the

quantity of DNA is limited (e.g., crime

scene sample). However, in most cases
41

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

involving high-throughput DNA typing (e.g., offender database work),

hundreds of nanograms of extracted

DNA would be easily available.

A major advantage of the mass spectrometry approach is speed of the

technique and the high-throughput

capabilities when combined with

robotic sample preparation. The data

collection times required for the 88

CDOJ samples using the ABI 310

Genetic Analyzer and GeneTrace’s

mass spectrometry method are compared in exhibit 11. While it took the

ABI 310 almost 3 days to collect the

data for the 88 samples, the same

genotypes were obtained on the mass

spectrometer in less than 2 hours.

Even the ability to analyze multiple

STR loci simultaneously with different

fluorescent tags on the ABI 310 could

not match the speed of GeneTrace’s

mass spectrometry data collection with

each marker run individually.

To verify that the mass spectrometry

approach produces accurate results,

comparison studies were performed on

the genotypes obtained from the two

different methods across 8 different

STR loci. Exhibits 12–19 contain a

direct comparison with 1,408 possible

data points (2 methods ✕ 88 samples

✕ 8 loci). With a few minor exceptions, there was almost a 100% correlation between the two methods. In

addition to the data obtained on the

8 loci from both the ABI 310 and the

mass spectrometer, two additional

markers (D8S1179 and DYS391) were

measured by mass spectrometry across

these same 88 samples (exhibits

39–40). Both the D8S1179 and the

DYS391 primer sets worked extremely well in the mass spectrometer

(exhibit 41). Thus, it is likely that if

results were made available on these

same samples with fluorescent STR

primer sets (e.g., D8S1179 is in the

AmpF1STR® Profiler PlusTM kit), there

would also be a further correlation

between the two methods.

PCR Issues

Null alleles

When making comparisons between

two methods that use different PCR

primer sets, the issue is whether or not

a different primer set for a given STR

locus will result in different allele calls

through possible sequence polymorphisms in the primer binding sites. In

other words, do primers used for mass

spectrometry that are closer to the

repeat region than those primers used

STEPS IN

PROCESS

 

Blood samples 

DNA FTA paper with blood spot

Extraction

Single punch Multiple punches

from the same spot

DNA (PCR ) Multiplex PCR with STR- Singleplex, duplex, or triplex

Amplification locus-specific primers PCR with GeneTrace

 (14 loci simultaneously) primers optimized for MS

 SPLIT SAMPLE

Second-round (nested PCR)

with GeneTrace primers

to probe 1-3 STRs per assay

Purification 384-well purification on robotic workstation

Mass Spec Automated mass spec detection

Detection

Data Analysis/ Automated genotyping at each locus

Genotyping

Databasing Data combined to form single sample genotype

Exhibit 38. Multiplexing strategies for STR genotyping using FTA paper TM
42

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

Exhibit 39. CDOJ D8S1179 STR results with the mass spectrometry method

Position Mass Spec Allele 1 (Da) Allele 2 (Da) Position Mass Spec Allele 1 (Da) Allele 2 (Da)

A1 12,15 24,693 28,228 A7 13,13 25,791

B1 14,15 27,159 28,369 B7 13,14 25,845 26,954

C1 11,14 23,342 26,933 C7 12,15 24,540 28,101

D1 15,15 28,199 D7 13,14 25,772 26,931

E1 11,14 23,421 26,988 E7 14,14 26,960

F1 13,17 25,822 30,529 F7 13,16 25,785 29,381

G1 11,14 23,449 27,093 G7 14,14 27,097

H1 14,14 27,067 H7 14,14 27,039

A2 13,13 25,777 A8 13,15 25,770 28,204

B2 12,14 24,597 27,007 B8 11,13 23,383 25,816

C2 11,11 23,361 C8 14,15 27,033 28,043

D2 11,13 23,399 25,768 D8 14,14 27,076

E2 13,14 25,904 27,012 E8 14,15 26,984 28,221

F2 14,14 27,125 F8 13,14 25,781 26,946

G2 No data G8 10,12 22,183 24,581

H2 11,15 23,344 28,151 H8 12,15 24,587 28,197

A3 15,15 28,160 A9 15,16 28,265 29,390

B3 13,15 25,904 28,256 B9 13,14 25,835 26,963

C3 12,14 24,640 27,022 C9 11,14 23,447 27,084

D3 11,15 23,407 28,201 D9 11,14 23,391 26,988

E3 11,14 23,461 27,093 E9 11,14 23,361 26,931

F3 14,15 27,086 28,186 F9 14,15 27,031 28,225

G3 10,15 22,218 28,230 G9 14,14 26,978

H3 13,16 25,849 29,355 H9 11,13 23,451 26,008

A4 14,15 27,018 28,112 A10 13,14 25,789 26,841

B4 12,14 24,605 27,018 B10 15,15 28,206

C4 13,13 25,797 C10 13,13 25,791

D4 No data D10 14,15 26,999 28,123

E4 14,14 27,108 E10 13,14 25,895 26,980

F4 14,15 27,080 28,215 F10 13,15 25,777 28,197

G4 12,13 24,311 25,519 G10 14,15 26,733 27,909

H4 13,14 25,824 27,044 H10 12,14 24,688 27,093

A5 12,16 24,548 29,321 A11 12,15 24,574 28,230

B5 13,14 25,893 26,997 B11 13,14 25,822 27,054

C5 12,13 24,727 25,885 C11 14,16 26,963 29,324

D5 No data D11 13,14 25,812 26,950

E5 14,14 26,960 E11 12,15 24,617 28,232

F5 13,14 25,916 27,014 F11 14,14 26,982

G5 14,15 27,009 28,106 G11 14,14 27,005

H5 13,14 25,804 26,708 H11 12,13 24,631 25,818

A6 14,14 26,988

B6 14,16 26,982 29,364

C6 15,15 28,249

D6 14,14 26,716

E6 13,16 25,933 29,584

F6 8,14 19,884 27,041

G6 15,15 28,394

H6 14,14 27,009
43

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

in fluorescent STR typing yield the

same genotype?

Differences between primer sets are

possible if there are sequence differences outside the repeat region that

occur in the primer binding region of

either set of primers (exhibit 42). This

phenomenon produces what is known

as a “null” allele, or in other words, the

DNA template exists for a particular

allele but fails to amplify during PCR

due to primer hybridization problems.

In all cases except the STR locus

D7S820, there was excellent correlation in genotype calls between the two

methods (where mass spectrometry

and CE results were obtained), signifying that the mass spectrometry primers

did not produce any null alleles. 

For the STR locus D7S820, 17 of 88

samples did not agree with the two

methods (exhibit 18). The bottom two

panels in exhibit 43 illustrate more

microheterogeneity at this locus than

previously reported. On the lower left

plot, only the allele 10 peak can be

seen; allele 8, which was seen with

PCR amplification using a fluorescent

primer set, is missing (see position of

red arrow in exhibit 43). On the lower

right plot, both allele 8 and allele 10

are amplified and detected in the mass

spectrometer, confirming that the problem is with the PCR amplification and

not the mass spectrometry data collection. In this particular case, there is a

difference between those two alleles 8,

meaning that the mass spectrometer

primer set identified a new, previously

unreported allele. When using fluorescent primer sets that anneal 50–100

bases or more from the repeat region,

a single-base change (e.g., T to C) out

of a 300 bp PCR product is difficult to

detect. Upon comparing the results of

mass spectrometer data where there

were missing alleles with the results

from the ABI 310, it was noted that

the situation occurred only with some

allele 8s, 9s, and 10s (see underlined

alleles in the ABI 310 column of

exhibit 18). Thus, these null alleles

were variants of alleles with 8, 9, or 

Exhibit 40. CDOJ DYS391 STR results with the mass spectrometry method

Position Mass Spec Allele 1 (Da) Position Mass Spec Allele 1 (Da)

A1 10 24,489 A7 10 24,416

B1 11 25,672 B7 10 24,406

C1 10 24,487 C7 10 24,353

D1 10 24,455 D7 8 21,905

E1 10 24,471 E7 11 25,662

F1 11 25,641 F7 10 24,353

G1 11 25,637 G7 10 24,422

H1 10 24,436 H7 10 24,451

A2 10 24,459 A8 10 24,455

B2 10 24,465 B8 11 25,529

C2 10 24,440 C8 10 24,410

D2 10 24,455 D8 10 24,473

E2 10 24,359 E8 12 26,841

F2 11 25,837 F8 10 24,438

G2 10 24,444 G8 10 24,359

H2 10 24,451 H8 12 26,805

A3 11 25,639 A9 10 24,367

B3 10 24,414 B9 10 24,416

C3 11 25,654 C9 10 24,463

D3 11 25,591 D9 10 24,343

E3 10 24,463 E9 10 24,471

F3 11 25,648 F9 10 24,457

G3 10 24,475 G9 10 24,465

H3 10 24,436 H9 10 24,479

A4 10 24,408 A10 11 25,631

B4 10 24,475 B10 10 24,617

C4 11 25,625 C10 10 24,560

D4 No data D10 10 24,444

E4 11 25,581 E10 11 25,650

F4 10 24,396 F10 10 24,414

G4 11 25,562 G10 10 24,453

H4 10 24,463 H10 11 25,866

A5 10 24,390 A11 11 25,662

B5 10 24,446 B11 10 24,432

C5 10 24,599 C11 10 24,400

D5 11 25,652 D11 10 24,463

E5 10 24,451 E11 10 24,420

F5 10 24,436 F11 9 23,175

G5 10 24,380 G11 10 24,459

H5 12 26,775 H11 11 25,585

A6 11 25,550

B6 10 24,428

C6 11 25,583

D6 10 24,457

E6 11 25,658

F6 10 24,436

G6 10 24,473

H6 10 24,463
44

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

10 repeats. Most likely, a sequence

microvariant occurs within the repeat

region near the 3’-end of the reverse

primer, which anneals to two full

repeats. Unfortunately, time constraints restricted the gathering of

sequence information for these samples to confirm the observed variation.

Interestingly enough, the D7S820

locus has been reported to cause similar null allele problems with other

primer sets (Schumm et al., 1997).

Microvariants

Sequence variation between alleles

can take the form of insertions, deletions, or nucleotide changes. Alleles

containing some form of sequence

variation compared with more commonly observed alleles are often

referred to as microvariants because

they are slightly different from full

repeat alleles. For example, the STR

locus TH01 contains a 9.3 allele, which

has 9 full repeats (AATG) and a partial repeat of 3 bases (ATG). In this

particular example, the 9.3 allele differs from the 10 allele by a single base

deletion of adenine. Microvariants

exist for most STR loci and are being

identified in greater numbers as more

samples are being examined around

the world. In this study, three previously unreported STR microvariants

(exhibit 44) were discovered during

the analysis of 38 genomic DNA samples from a male population data set

provided by Dr. Oefner (exhibit 45).

These microvariants occurred in the

three most polymorphic STR loci that

possess the largest and most complex

repeat structures: FGA, D21S11, and

D18S51.

The ability to make accurate mass

measurements with mass spectrometry is a potential advantage when

locating new microvariants. If the

mass precision is good, then any

peaks that have large offsets from the

expected full repeat alleles could be

suspect microvariants in the form of

insertions or deletions because their

masses would fall outside the expected

variance due to instrument variation.

This possibility is especially true when

working with heterozygous samples.

Microvariants can be detected by using

the mass difference between the two

alleles and comparing this value with

the expected value for full repeats or

with the allele peak mass offsets. If

the peak mass offsets shift together,

then both alleles are full repeats, but

if one of the peak mass offsets is significantly different (e.g., ~300 Da), a

possible insertion or deletion exists

in one of the alleles. Exhibit 46 illustrates this concept by plotting the mass

offset (from a calculated allele mass)

of allele 1 verses the mass offset (from

a calculated allele mass) of allele 2.

Note that the 9.3 microvariant (i.e.,

partial) repeat alleles for TH01 cluster away from the comparison of full

repeat versus full repeat allele. On 

the other hand, results for the other

three STR loci, which have no known

microvariants in this data set, have

mass offsets that shift together for the

heterozygous alleles. Exhibit 47 compares the peak mass offsets for the

amelogenin X allele with the Y allele

and demonstrates that full “repeats”

shift together during mass spectrometry measurements.

Nontemplate addition

DNA polymerases, particularly the

Taq polymerase used in PCR, often

add an extra nucleotide to the 3’-end

of a PCR product as template strands

are copied. This nontemplate addition—which is most often an adenine,

hence, the term “adenylation”—can 

be favored by adding a final incubation step at 60 o

C or 72 oC after the

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

55,000

0 96 192 288 384

Mass (Da)

FGA D8S1179 DYS391

9 repeats

20 repeats

16 repeats

30 repeats

8 repeats

15 repeats

8 repeats

12 repeats

CDOJ Samples Plate 970805A

Sample number

D3S1358

Exhibit 41. Plot of measured masses versus sample number from four 

different STR loci. FGA, which has a high degree of scatter, is the most 

polymorphic marker and has the largest mass alleles. The highest and lowest 

alleles observed for each STR locus in this study are shown on the plot.
45

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

temperature cycling steps in PCR

(Clark, 1988, and Kimpton et al.,

1993). However, the degree of adenylation is dependent on the sequence

of the template strand, which in the

case of PCR results from the 5’-end 

of the reverse primer. Thus, every

locus will have different adenylation

properties because the primer

sequences are different. From a measurement standpoint, it is better to have

all molecules of a PCR product as similar as possible for a particular allele.

Partial adenylation, where some of the

PCR products do not have the extra

adenine (i.e., -A peaks) and some do

(i.e., +A peaks), can contribute to peak

broadness if the separation system’s

resolution is poor (see top panel of

exhibit 37). Sharper peaks improve the

likelihood that a system’s genotyping

software can make accurate calls.

Variation in the adenylation status of

an allele across multiple samples can

have an impact on accurate sizing and

genotyping potential microvariants.

For example, a nonadenylated TH01

10 allele would look the same as a

fully adenylated TH01 9.3 allele in

the mass spectrometer because their

masses are identical. Therefore, it is

beneficial if all PCR products for a

particular amplification are either +A

or -A rather than a mixture (e.g., ±A).

By using the temperature soak at the

end of thermal cycling, most of the

STR loci were fully adenylated, with

the notable exception of TPOX, which

was typically nonadenylated, and

TH01, which under some PCR conditions produced partially adenylated

amplicons. For making correct genotype calls, the STR mass ladder file

(exhibit 30) was altered according to

the empirically determined adenylation status.

During the course of this project,

Platinum® GenoTYPETM Tsp DNA polymerase (Life Technologies, Rockville,

MD) became available that exhibits

little to no nontemplate nucleotide

addition. This new DNA polymerase

was tested with STR loci that had been

shown to produce partial adenylation

to see if the +A peak could be eliminated. Exhibit 48 compares mass

spectrometry results obtained using

AmpliTaq Gold (commonly used)

polymerase with the new Tsp polymerase. The Tsp polymerase produced

amplicons with only the -A peaks,

while TaqGold showed partial adenylation with these TH01 primers. Thus,

this new polymerase has the potential

to produce sharper peaks (i.e., no

partial adenylation) and allele masses 

that can be more easily predicted 

*

*

*

(A)

(B)

(C)

Exhibit 42. Effects of sequence variation on PCR amplification in or 

around STR repeat regions. The asterisk symbolizes a DNA difference (base 

change, insertion, or deletion of a nucleotide) from a typical allele for a STR 

locus. In situation (A), the variation occurs within the repeat region (depicted 

in green) and should have no impact on the primer binding and the subsequent 

PCR amplification, although the overall amplicon size may vary slightly. In 

situation (B), the sequence variation occurs just outside the repeat in the 

flanking region but interior to the primer annealing sites. Again, PCR should 

not be affected, although the size of the PCR product may vary slightly. 

However, in situation (C), the PCR can fail due to a disruption in annealing a 

primer because the primer no longer perfectly matches the DNA template 

sequence. Therefore, if sequence variation occurs in the flanking region for a 

particular locus, one set of primers may work while another may fail to amplify 

the template. The template would therefore be a “null” allele.
46

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

(i.e., all PCR products would be 

nonadenylated).

Stutter products

During PCR amplification of STR loci,

repeat slippage can occur and result in

the loss of a repeat unit as DNA strand

synthesis occurs through a repeated

sequence. These stutter products are

typically 4 bases, or one tetranucleotide

repeat, shorter than the true allele PCR

product. The amount of stutter product

compared to the allele product varies

depending on the STR locus and the

length of the repeat, but typically stutter peaks are 2–10% of the allele peak

height (Walsh et al., 1996). Forensic

DNA scientists are concerned about

stutter products because their presence

can interfere in the interpretation of

DNA mixture profiles.

When reviewing plots of GeneTrace’s

mass spectrometry results for STR loci,

forensic scientists have commented on

the reduced level of stutter product

Exhibit 43. Mass spectra of CDOJ samples amplified with D7S820 primers. From left side, top-to-bottom followed 

by right side, top-to-bottom, sample genotypes are (10,11), (9,10), (10,10), (null allele 8, 10), (8,11), (11,13), (10,12), and

(8,10). The arrow indicates the position where allele 8 should be present in the sample but is missing due to a primer 

annealing binding site sequence polymorphism that results in a “null allele.” The mass range shown here is 

12,000–25,000 Da. 

1.5

 x 104

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

2 2.5 1.5 2 2.5

1.5 2 2.5

 x 104 x 104

Intensity (arbitrary units) Intensity (arbitrary units) Intensity (arbitrary units) Intensity (arbitrary units)

Intensity (arbitrary units) Intensity (arbitrary units)

Intensity (arbitrary units) Intensity (arbitrary units)

10,000

0

2

0

5,000 1

1.5 2 2.5 1.5 2 2.5

10,000

0

5,000

1.5 2 2.5

1.5 2 2.5 1.5 2 2.5

10,000

0

5,000 10,000

5,000

0

2

0

1

0 0

 x 104

10,000

10,000
47

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

detection (exhibit 35). There are two

possibilities for this reduction:

◆ Since the primers are closer to the

repeat region, smaller PCR products

are amplified, which means that the

DNA polymerase does not have to

hold on to the extending strand as

long for synthesis purposes. It is

possible that the polymerase reads

through the repeat region “faster”

and, therefore, the template strands

do not have as much of an opportunity to slip and reanneal out of

register on the repeat region. For

example, Taq polymerase has a processivity rate of ~60 bases before it

falls off the extending DNA strand;

therefore, the closer the PCR product size is to 60 bases, the better the

extension portion of the PCR cycle.

GeneTrace’s PCR product sizes,

which are typically less than 100

bp, are much smaller than the

1,500

Exhibit 44. Electropherograms of ABI 310 results for new STR microvariants seen in the Stanford male population 

samples. The D18S51 16.2 allele, D21S11 30.3 allele, and FGA 28.1 allele have not been reported previously in the 

literature. These plots are views from Genotyper 2.0 with results overlaid on shaded allele bins. The base pair size range is 

indicated at the top of each plot. Note: The microvariant alleles (indicated by the red arrows) fall between the shaded bins, 

but the other alleles in the heterozygote set contain complete repeats and fall directly on the shaded (expected) allele bin. 

2,000

1,000

500

2,000

3,000

1,000

3,000

4,000

2,000

1,000
48RESULTS AND

DISCUSSION OF STR ANALYSIS BYMASSSPECTROMETRY

Name

Sample AmelA1 Amel- A2 D8- A1 D8- A2 D21-A1 D21- A2 D18- A1 D18 -A2 D3- A1 D3- A2 VWA- A1 VWA -A2 FGA- A1 FGA- A2 D5- A1 D5- A2 D13- A1 D13 -A2 D7- A1 D7- A2

Aus21 X Y 8 12 29 31.2 12 18 15 17 14 17 18 22 12 12 11 12 9 11Aus28 X Y 14 15 29 31.2 15 19 16 17 15 17 19 20 12 12 10 12 8 10Berg15 X Y 10 28,30.2 31.2 12,17 14 15,17 18,19 16 17,18 20,21 24 11 13w 8,14 11 7w 10Berg19 X Y 14 14 32.2 32.2 12 18 16 16 14 18 22 22 12 13 12 12 9 13BI12 X Y 12 12 30 31.2 17 17 16 18 17 19 24 27 12 12 11 11 9 11Bsk092 X Y 14 14 29 33.2 13 16 17 17 17 17 23 23 11 12 12 12 11 11Bsk111 X Y 12 13 30 32.2 16 19 14 14 16 16 18 19 12 12 8 10 8 9Bsk118 X Y 13 14 30.2 30.2 11 14 16 16 17 17 23 25 10 12 8 10 8 8CH17 X Y 12 14 32.2 34.2 14 23 15 15 16 18 20 26 9 12 8 12 11 12CH23 X Y 12 13 29 31 16 16 15 15 14 17 24 24 11 12 9 12 10 12CH42 X Y 12 14 28.2 31.2 13 15 16 16 17 17 21 22 12 12 10 12 11 11F18 X Y 14 14 31 32.2 19 19 15 15 14 16 21 23 13 13 11 12 8 12F21 X Y 14 15 27 31 14 19 15 15 17 18 22 26 12 13 11 13 8 8J13 X Y 14 14 32.2 33.2 17 19 15 17 14 16 20 22 10 11 8 11 10 10J3 X Y 13 14 30 30 13 15 16 16 17 17 22 24 10 14 8 11 10 10J37 X Y 10 13 31 31.2 13 20 15 15 16 18 22 25 10 12 8 11 10 10J39 X Y 13 14 30 31.2 13 13 15 18 17 17 23 24 11 13 12 13 8 12JK2921 X Y 10 10 29 31.2 14 15 15 16 14 19w 19 22 10 11 11 11 9 9JK2979 X Y 10 12 32.2 33.2 14 14 15 16 14 18 21 24.2 11 14 11 12 11 11MDK204 X Y 14 17 28 30.2 12 19 14 15 16 16 23 25 12 13 11 12 8 10Mel12 X Y 13 14 30 32.2 17 20 15 16 17 18 21 22 13 14,15 11 12 12 12

Mel15 X Y 14 15 28 31.2 14,15 22 16 18 16 18 23 24 10 10 12 13 11 11

Mel18 X Y 15 15 28 32.2 14 15 15 16 17 18 24 24 10 11 12 13 10 11

NG 83 X Y 13 13 30 31 1 3 18 15 16 1 5 1 7 23 24 11 11 8 8 8 10

NG85 X Y 15 15 29 31.2 15 21 16 17 17 19 23 26 13 13 8 8 11 12NG88 X Y 12 15 33.2 38.2 13 17 15 15 16 16 25 28.1 11 11 8 11 8 10

OM135 X Y 12 13 28 39 15 16 16 16 16 18 21 25 12 12 12 12 11 11P103G X X 12 17 29 30 19 20 17 17 15 19 20 24 13 13 10 12 8 8

P109 X Y 11 14 28 34 17 21 15 16 15 19 22 27 11 12 12 12 9 10P205 X Y 11 15 28 36 15 15 14 15 15 16 21 24 8 10 12 12 9 10P240 X Y 14 14 30.3 34 16 16 16 17 15 21 22 22.2 10 11 8 13 11 12

P33 X Y 13 14 29 30 16.2 18 14 17 16 17 20 24 8 10 11 12 10 10

P37G X Y 14 15 29 32.2 14 20 16 17 17 18 24 24 12 13 9 12 8 11P37G? X Y 14 15 29 32.2 14 20 16 17 17 18 24 24 12 13 9 12 8 11P73 X Y 14 15 29 30 14 16 15 16 14 19 22.2 24 12 12 11 13 10 11PG1162 X Y 8 14 31.2 32.2 14 15 15 16 16 18 18 23 11 12 9 11 8 10PKH062 X Y 12 15 29 31 13 16 16 18 16 18 21 22 11 11 9 13 10 12SDH053 X Y 10 12 30 33.2 16 16 18 18 17 17 21 25 11 12 8 8 8 8

Exhibit 45. ProfilerPlusTM results from Stanford male population samples 12,13

Note: The STR loci are color coded to indicate their fluorescent dye label color; the shaded boxes in the body of the exhibit refer to the microvariant alleles 

(exhibit 44), 3-banded patterns, or an unexpected “x,x” amelogenin.
49

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

fluorescently labeled primer sets

used by most forensic DNA laboratories (exhibit 2). However, this

needs to be studied more extensively with multiple primer sets on a

particular STR locus that generates

various sized amplicons. For example, the primer sets described in

exhibit 24 could be fluorescently

labeled and analyzed on the ABI

310, where the stutter product peak

heights could be quantitatively

compared to the allele peak heights.

◆ The more likely reason that less

stutter is observed by mass spectrometry is that the signal-to-noise

ratio is much lower in mass spectrometry than in fluorescence

measurements. Fluorescence techniques have a much lower background and are more sensitive for

the detection of DNA than mass

spectrometry. Thus, stutter may be

present at similar ratios compared

with those observed in fluorescence

measurements, but because stutter

is part of the baseline noise of mass

spectrometry data, it may not be

seen in the mass spectrum. This

latter explanation is probably more

likely, as indicated in very strong

stutter peaks for some dinucleotide

repeat markers (exhibit 49).

Whether stutter products are present

or not, GeneTrace’s current STR genotyping software has been designed

to recognize them and not call them

as alleles.

Primer sequence 

determinations from 

commercial STR kits

Primarily, two commercial manufacturers supply STR kits to the forensic

DNA community: Promega Corporation and Applied Biosystems. These

kits come with PCR primer sequences

that permit simultaneous multiplex

PCR amplification of up to 16 STR

loci. One of the primers for each STR

locus is labeled with a fluorescent

-800

-600

-400

-200

0

200

400

600

800

0 200 400 600

Mass offset 2 (Da)

300 Da window

(1 base)

 100 Da window 

Microvariants

-600 -400 -200

Mass offset 1 (Da)

Exhibit 46. Plot of allele mass offsets (allele 1 versus allele 2) for 

heterozygous samples from four different loci. These are 88 CDOJ 

samples for the STR loci TH01, TPOX, CSF1PO, and D16S539.

Exhibit 47. Plot of X allele mass offset versus Y allele mass offset for 88 

amelogenin samples. The red box shows ±100 Da around the expected values 

and the green box shows ±300 Da. The blue line is the ideal situation where 

“heterozygous” peaks would shift in unison compared with the expected 

masses. 

-600

0

600

600

Mass offset Y allele (Da)

300

300

-300

-300 -300

Mass offset X allele (Da)

0
50

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

dye to permit fluorescent detection of

the labeled PCR products. Since the

primer sequences are not disclosed

by the manufacturers, mass spectrometry was used to determine where they

annealed to the STR sequences compared with GeneTrace primers (see

previous discussion on null alleles).

First, the primer mixtures were spotted and analyzed to determine each

primer’s mass (top panel of exhibit

50). Then a 5’→3’ exonuclease was

added to the primer mix and heated to

37 oC for several minutes to digest the

primer one base at a time. An aliquot

was removed every 5–10 minutes to

obtain a time course on the digestion

reaction. Each aliquot was spotted in

3-hydroxypicolinic acid matrix solution (Wu et al., 1993), allowed to dry,

and analyzed in the mass spectrometer.

A digestion reaction produces a series

of products that differ by one nucleotide. By measuring the mass difference

between each peak, the original primer

sequence may be determined (bottom

panel of exhibit 50). Only the unlabeled primers will be digested because

the covalently attached fluorescent dye

blocks the 5’-end of the dye-labeled

primer. Using only a few bases of

sequence (e.g., 4–5 bases), it is possible

to make a match on the appropriate

STR sequence obtained from GenBank

to determine the 5’-end of the primer

without the fluorescent label.

With the full-length primer mass

obtained from the first experiment, 

the remainder of the unlabeled primer

can be identified. The position of the

5’-end of the other primer can be determined using the GenBank sequence

and the PCR product length for the

appropriate STR allele listed in GenBank

(exhibit 2). The sequence of the labeled

primer can be ascertained by using the

appropriate primer mass determined

from the first experiment and subtracting the mass of the fluorescent dye.

The primer mass is then used to obtain

the correct length of the primer on the

GenBank sequence and the primer’s

Exhibit 48. Mass spectra comparing an STR sample amplified with 

TaqGold polymerase and Tsp polymerase. The Tsp polymerase favors 

production of the nonadenylated form of PCR products, which results in a single 

peak for each allele (bottom panel). TaqGold produces a mixture of –A and +A 

peaks, which leads to two peaks for each allele (top panel). The peak masses in 

Daltons are indicated next to each peak. Mass difference measurements of 308 

Da and 305 Da between the +A and –A peaks reveal that a “T” is added by 

TaqGold instead of the expected “A” (expected masses: T = 304 Da and

A = 313 Da). The sample’s genotype was TH01 6,8. 

14,895

15,203

17,706

17,421

14,907

Tsp polymerase 

(only -A)

TaqGold polymerase 

(only ±A)

12,000 14,000 16,000 18,000 20,000

17,401

Mass (Da)

Signal

Exhibit 49. Mass spectrum demonstrating detection of stutter products 

from a particularly stutter-prone dinucleotide repeat locus. The mass 

differences between the stutter product peaks and the allele peaks can be used 

to determine the repeat sequence that is present on the measured DNA strand. 

Note: The amount of stutter is larger in the longer repeat allele than in the 

shorter allele.

14,000 16,000 18,000 20,000 22,000

Mass (Da)

Signal

CT repeat = 593 Da

13 repeats

18 repeats

∆m = 592

∆m = 587

∆m = 590

∆m = 590

∆m = 587
51

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

sequence. Finally, an entire STR

multiplex primer set can be measured

together in the mass spectrometer to

observe the primer balance (exhibit

51). High-performance liquid chromatography fraction collection can be

used to pull primers apart from complex, multiplex mixtures, and each

primer can be identified as previously

described. The primer sequences from

both Promega and Applied Biosystems

STR loci TH01 (exhibit 52), TPOX

(exhibit 53), and CSF1PO (exhibit 54)

were identified using this procedure.

A comparison of the primer sequences

from the two manufacturers found that

they were very similar. The 3’-ends

of the primer sets—the most critical

portions for annealing during—were

almost identical between the different

kits. The ABI primers were typically

shorter at the 5’-end and, therefore,

produced PCR products that were ~10

bases shorter than those produced by

the corresponding Promega primers.

In all three STR loci, the primers

annealed further away from the repeat

region than the GeneTrace primer sets.

Analytical Capabilities

of This Mass Spectrometry Method

Using the current primer design strategy, most STR alleles ranged in size

from ~10,000 Da to ~40,000 Da. In

mass spectrometry, the smaller the

molecule, the easier it is to ionize and

detect (all other things being equal).

Resolution, sensitivity, and accuracy

are usually better the smaller the DNA

molecule being measured. Because the

possible STR alleles are relatively far

apart, reliable genotyping is readily

attainable even with DNA molecules

at the higher mass region of the spectrum. For example, neighboring fulllength alleles for a tetranucleotide

repeat, such as AATG, differ in mass

by 1,260 Da.

5,000 6,000 7,000 8,000 9,000

Dye-labeled

primer

Original primer pair

(not desalted)

Use of mass spectrometry

to determine PCR primers

of unknown sequence

Following 6 min digestion of

primers with 5’ 3’ exonuclease

Exhibit 50. Primer sequence determination with exonuclease digestion 

and mass difference measurements. This example is a D5S818 primer pair 

purchased from Promega Corporation and used in its PowerPlexTM STR 

kit. The top panel shows a mass spectrum of the original primer pair prior to 

digestion. The bottom panel is the mass spectrum of the same primers following 

a 6 minute digestion at 37 oC with calf spleen phosphodiesterase, which is a 

5’→3’ exonuclease. The dye-labeled primer is not digested because the dye 

protects the 5’-end of the primer. Mass difference measurements between 

the digestion peaks leads to the sequence determination of the 5’-end of the 

unlabeled primer (see underlined portion of forward sequence). The determined 

sequences are 5’-GGTGATTTTCCTCTTTGGTATCC-3’ (forward) and 

5’-fluorescein dye—TTTACAACATTTGTATCTATATCTGT-3’ (reverse).

G G

G T T

T T

A

→

Signal

Mass (Da)

CSF-R

TH01-F

TPOX-R

TPOX-F

CSF-F

TH01-R

AMEL-R

AMEL-F

5,000 6,000 7,000 8,000

Exhibit 51. Mass spectrum of AmpF1STR® Green I primer mix. Each 

peak has been identified with its corresponding primer. Peaks containing 

the fluorescent dye (JOE) are underlined.

Mass (Da)

Signal
52

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

AmpFlSTR® Green I Kit 

Reverse primer is labeled with JOE dye (fluorescein derivative) 

 PCR product =184 bp (9 repeats) 

PowerPlexTM Kit

Forward primer is labeled with TMR dye (tetramethylrhodamine) 

PCR product = 195 bp (9 repeats)

Exhibit 52. TH01 STR primer positions for commercially available primers highlighted on the GenBank sequence. 

The forward primer is shown in blue with the reverse primer in brown. The repeat is highlighted in green on the strand 

that contains the fluorescent dye. The 3’-positions of the forward primers are identical but differ by a single base for the 

reverse primers. Promega primers are longer at the 5’-end, which produces a larger PCR product by 11 bp (6 bases on 

forward and 5 bases on reverse). 
53

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

AmpFlSTR® Green I Kit

Forward primer is labeled with JOE dye (fluorescein derivative) 

PCR product = 237 bp (11 repeats) 

PowerPlexTM Kit

Reverse primer is labeled with TMR dye (tetramethylrhodamine) 

PCR product = 244 bp (11 repeats) 

Exhibit 53. TPOX STR primer positions for commercially available primers highlighted on the GenBank 

sequence. The forward primer is shown in blue with the reverse primer in brown. The repeat is highlighted in green on 

the strand that contains the fluorescent dye. The 3’-positions of the reverse primers are identical but differ by a single 

base for the forward primers. Promega primers are longer at the 5’-end, which produces a larger PCR product by 7 bp 

(4 bases on forward and 3 bases on reverse).
54

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

AmpFlSTR® Green I Kit

Forward primer is labeled with JOE dye (fluorescein derivative) 

PCR product = 304 bp (12 repeats) 

PowerPlexTM Kit 

Forward primer is labeled with TMR dye (tetramethylrhodamine)

PCR product = 315 bp (12 repeats) 

Exhibit 54. CSF1PO STR primer positions for commercially available primers highlighted on the GenBank 

sequence. The forward primer is shown in blue with the reverse primer in brown. The repeat is highlighted in green on the 

strand that contains the fluorescent dye. The 3’-positions of the reverse primers are identical but differ by a single base for 

the forward primers. Promega primers are longer at the 5’-end, which produces a larger PCR product by 11 bp (5 bases on 

forward and 6 bases on reverse). 
55

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

Resolution

Dinucleotide repeats, such as CA

repeats, require a resolution of at least

2 bp in order to resolve stutter products from the true allele or heterozygotes that differ by a single repeat.

Trinucleotide and tetranucleotide

repeats, with their larger repeat structure, are more easily resolved because

there is a larger mass difference

between adjacent alleles. However,

the overall mass of the PCR product

increases more rapidly with tri- or

tetranucleotide repeats. For example,

the repeat region for 40 GA repeats

is 25,680 Da, while the mass of the

repeat region quickly increases to

37,200 Da for 40 AAT repeats and

50,400 Da for 40 AATG repeats.

GeneTrace has demonstrated that a

resolution of a single dinucleotide

repeat (~600 Da) may be obtained

for DNA molecules up to a mass of

~35,000 Da. This reduced resolution

at higher mass presents a problem for

polymorphic STR loci such as D18S51,

D21S11, and FGA because single base

resolution is often required to accurately call closely spaced alleles or to

distinguish a microvariant containing a

partial repeat from a full-length allele.

These three STR loci also contain 

long alleles. For example, D21S11 has

reported alleles of up to 38 repeats

(mixture of TCTA and TCTG) in

length, D18S51 up to 27 AGAA

repeats, and FGA up to 50 repeats

(mixture of CTTT and CTTC).

Heterozygous FGA alleles that differed

by only a single repeat were more difficult to genotype accurately than

smaller sized STR loci due to poor

resolution at masses greater than

~35,000 Da (see samples marked in

red in exhibit 19).

The analysis of STR allelic ladders

demonstrates that all alleles can be

resolved for an STR locus. Allelic ladders from commercial kits were typically diluted 1:1000 with deionized

water and then reamplified with the

Exhibit 55. Mass spectrum of a TH01 allelic ladder reamplified from 

AmpF1STR® Green I allelic ladders. The PCR product size of allele 10 is only 

83 bp with a measured mass of 20,280 Da and separation time of 204 μs. The 

allele 9.3 and allele 10 peaks, which are only a single nucleotide apart, differ by 

only 1.5 μs on a separation timescale and can be fully resolved with this method. 

14,000 16,000 18,000 20,000

m/z

5 6 7

8

9

9.3

10

Signal

Exhibit 56. Mass spectra of D5S818 allelic ladders from two manufacturers.

The Applied Biosystems D5S818 ladder contains 10 alleles (top panel); the 

Promega D5S818 ladder contains only 8 alleles (bottom panel). The GeneTrace 

primers bind internally to commercially available multiplex primers, and all 

alleles in the commercial allelic ladders are therefore amplified, demonstrating 

that the GeneTrace primers can amplify all common alleles for this particular 

STR locus. 

14,000 16,000 18,000 20,000 22,000 24,000 26,000 28,000 Signal

Mass (Da)

D5S818 Allelic Ladder

from Promega

D5S818 Allelic Ladder from 

Applied Biosystems

7

8 9

10

11

12 13 14

15

16

7

8 9 10 11

12 13

14
56

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

GeneTrace primers that bound closer

to the repeat region than the primers

from the commercial kits. This reamplification provided PCR products for

demonstrating that the needed level of

resolution (i.e., distinguishing adjacent

alleles) is capable at the appropriate

mass range in the mass spectrometer

as well as demonstrating that the

GeneTrace primers amplify all alleles

(i.e., no allele dropout from a null

allele). A number of STR allelic ladders

were tested in this fashion, including

TH01 (exhibit 55); CSF1PO, TPOX,

and VWA (exhibit 36); and D5S818

(exhibit 56). All tetranucleotide repeat

alleles were resolvable in these examples, demonstrating 4 bp resolution,

and TH01 single base pair resolution

was seen between alleles 9.3 and 10.

Sensitivity

To determine the sensitivity of GeneTrace’s STR typing assay, TPOX

primers were tested with a dilution

series of K562 genomic DNA (20 ng,

10 ng, 5 ng, 2 ng, 1 ng, 0.5 ng, 0.2 ng,

and 0 ng). Promega’s Taq polymerase

and STR buffer were used with 35

PCR cycles as described in the scope

and methodology section. Peaks for

the correct genotype (heterozygote

8,9) could be seen down to the lowest

level tested (0.2 ng or 200 picograms),

while the negative control was blank.

Exhibit 57 contains a plot with the

mass spectra for 20 ng, 5 ng, 0.5 ng,

and 0 ng. While each PCR primer pair

can exhibit a slightly different efficiency, human DNA down to a level of ~1

ng can be reliably PCR amplified and

detected using mass spectrometry.

GeneTrace’s most recent protocol

involved 40-cycle PCR and the use of

TaqGoldTM DNA polymerase, which

should improve overall yield for STR

amplicons. All of the samples tested

from CDOJ were amplified with only

5 ng of DNA template and yielded

excellent results (exhibits 12–19, 31,

39, 40, 43, 58, and 59). In terms of

absolute sensitivity in the mass spectrometer, several hundred femtomoles

of relatively salt-free DNA molecules

were typically found necessary for

detection. GeneTrace’s PCR amplifications normally produced several picomoles of PCR product, approximately

an order of magnitude more material

than is actually needed for detection.

Mass accuracy and 

precision

Mass accuracy is an important issue

for this mass spectrometry approach to

STR genotyping, as a measured mass

for a particular allele is compared with

an ideal mass for that allele. Due to

Exhibit 57. Mass spectra of TPOX PCR products from various amounts of 

K562 DNA template material. This sensitivity test demonstrates that DNA 

templates in the quantity range of 0.5–20 ng may be effectively amplified and 

detected by mass spectrometry. 

14,000 16,000 18,000 20,000 22,000 24,000

9

Signal

8

9 8

20 ng K562

5 ng K562

9

8

0.5 ng K562 primer

dimer

0 ng (negative control)

Mass (Da)
57

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

the excellent accuracy of mass spectrometry, internal standards are not

required to obtain accurate DNA sizing results as in gel or CE measurements (Butler et al., 1998). To make

an inaccurate genotype call for a

tetranucleotide repeat, the mass offset

from an expected allele mass would

have to be larger than 600 Da (half 

the mass of a ~1,200 Da repeat).

GeneTrace has observed mass accuracies on the order of 0.01 nucleotides

(<3 Da) for STR allele measurements.

However, under routine operation

with GeneTrace’s automated mass

spectrometers, some resolution, sensitivity, and accuracy may be sacrificed

compared with a research-grade instrument to deliver data at a high rate of

speed. Almost all STR allele size measurements should be within ±200 Da,

or a fraction of a single nucleotide, of

the expected mass. Exhibit 60 illustrates that the precision and accuracy

for STR measurements is good enough

to make accurate genotyping calls with

only a routine mass calibration, even

when comparing data from the same

samples collected months apart.

Exhibit 58. Mass spectra of CDOJ samples amplified with TPOX primers. From left side, top-to-bottom, followed by 

right side, top-to-bottom, sample genotypes are (8,9), (9,10), (10,11), (11,12), (8,8), (8,11), (11,11), and allelic ladder. The 

mass range shown here is 15,000–25,000 Da.

1.5

 x 104

 x 104

 x 104

Mass (Da)

Intensity (arbitrary units)

Mass (Da)

Intensity (arbitrary units)

Mass (Da)

Mass (Da)

Intensity (arbitrary units) Intensity (arbitrary units)

Intensity (arbitrary units) Intensity (arbitrary units) Intensity (arbitrary units) Intensity (arbitrary units)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

2 2.5 1.5 2 2.5

1.5 2 2.5 1.5 2 2.5

1.5 2 2.5 1.5 2 2.5

1.5 2 2.5 1.5 2 2.5

5

 x 104

 x 104

 x 104

 x 104

10

 x 104

0

0

5

10

0

5

0

5

10

0

0

5

0

5

10

5

0

 x 104 x 104

10
58

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

Precision is important for STR allele

measurements in mass spectrometry

because no internal standards are

being run with each sample to make

adjustments for slight variations in

instrument conditions between runs.

To demonstrate the excellent reproducibility of mass spectrometry, 15

mass spectra of a TPOX allelic ladder

were collected. A table of the obtained

masses for alleles 6, 7, 8, 9, 10, 11, 12,

and 13 shows that all alleles were

easily segregated and distinguishable

(exhibit 61). Statistical analysis of the

data found that the standard deviation

about the mean for each allele ranged

from 20 to 27 Da, or approximately

0.1% relative standard deviation (RSD).

The mass between alleles is equal to

the repeat unit, which in the case of

TPOX is 1,260 Da for an AATG repeat

(exhibit 62). Thus, each allele is easily

distinguishable. 

Measurements were made of the same

DNA samples over a fairly wide timespan, revealing that masses can be

remarkably similar, even when data

points are recollected months later.

Exhibit 60 compares 57 allele measExhibit 59. Mass spectra of CDOJ samples amplified with TH01 primers. From left side, top-to-bottom followed by right 

side, top-to-bottom, sample genotypes are (6,7), (6,8), (6,9.3), (6,10), (7,7), (7,7), (7,8), and (7,9.3). The split peaks for each 

allele result from partial adenylation—i.e., both ±A peaks are present. The mass range shown here is 12,000–22,000 Da. 

1.2

 x 104

 x 104

 x 104

Mass (Da)

Intensity (arbitrary units)

Mass (Da)

Intensity (arbitrary units)

Mass (Da)

Mass (Da)

Intensity (arbitrary units) Intensity (arbitrary units)

Intensity (arbitrary units) Intensity (arbitrary units) Intensity (arbitrary units) Intensity (arbitrary units)

Mass (Da)

Mass (Da)

Mass (Da)

Mass (Da)

1.4

1

 x 104

 x 104

x 104

x 104

2

x 104

0

0

5

0

1

2

0

0

1

0

2

5

0

 x 104 x 104

2

1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

1.2 1.4 1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

1.2 1.4 1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

1.2 1.4 1.6 1.8 2.0 2.2 1.2 1.4 1.6 1.8 2.0 2.2

0

1

2

2
59

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

urements from 6 different TPOX alleles collected 6 months apart. The first

data set was collected on October 1,

1998, and the second data set on

March 26, 1999. Amazingly enough,

some of the alleles had identical measured masses, even though different

mass calibration constants (and even

different instruments) were used.

The bottom line is whether or not a

correct genotype can be obtained using

this new technology. Exhibit 63 compares the genotypes obtained using a

conventional CE separation method

and this mass spectrometry technique

across 3 STR markers (D16S539,

D8S1179, and CSF1PO) and indicates

an excellent agreement between the

methods. With the CDOJ samples tested, there was complete agreement on

all observed genotypes for the STR loci

CSF1PO, TH01, and D3S1358 as well

as the sex-typing marker amelogenin

(exhibits 12, 14–16). Some “gas-phase”

dimers and trimers fell into the allele

mass range and confused the calling

for TPOX (exhibit 13) and D16S539

(exhibit 17) on several samples. Gasphase dimers and trimers are assay

artifacts that result from multiple

excess primer molecules colliding in

the gas phase and being ionized during the MALDI process. A mass offset

plot like that shown in exhibit 46 can

be used to detect these assay artifacts

as they fall outside the tight grouping

and inside the 300 Da window. With

the CDOJ samples, D7S820 exhibited

null alleles (exhibit 18) and FGA had

some unique challenges due to its larger size, such as problems with resolution of closely spaced heterozygotes

and poorer mass calibration since the

measured alleles were further away

from the calibration standards (exhibit

19). Thus, when the PCR situations

such as null alleles are accounted for

and smaller loci are used, this mass

spectrometry method produces results

comparable to traditional methods of

STR genotyping.

Data collection speed

The tremendous speed advantage of

mass spectrometry can be seen in

exhibit 11. Over the course of this

project, data collection speed increased

by a factor of 10 from ~50 seconds/

sample to less than 5 seconds/sample.

This speed increase resulted from

improved software and hardware on

the automated mass spectrometers

and from improved sample quality

(i.e., better PCR conditions that yielded more product and improved sample

cleanup that in turn yielded “cleaner”

DNA). With data collection time around

5 seconds per sample, achieving sample throughputs of almost 1,000 samples per hour is possible, and 3,000–

4,000 samples per system per day is

reasonable when operating at full

capacity. Sample backlogs could be

erased rather rapidly with this kind of

throughput. By way of comparison, it

takes an average of 5 minutes to obtain

each genotype (assuming a multiplex

level of 6 or 7 STRs) using conventional CE methods (exhibit 11).

Thus, the mass spectrometry method

described in this study is two orders of

magnitude faster in sample processing

time than conventional techniques.

24,000

15,000 16,000 17,000 18,000 19,000 20,000 21,000 22,000 23,000 24,000

Observed mass (Da) (Mar. 26, 1999)

23,000

22,000

21,000

20,000

19,000

18,000

17,000

16,000

15,000

Observed mass (Da) (Oct. 1, 1998)

Exhibit 60. Comparison of allele masses collected 6 months apart. This plot 

compares 57 allele measurements of 6 different TPOX alleles. The ideal line is 

shown on the same plot to demonstrate how reproducible the masses are over 

time. The average standard deviation of allele mass measurements between 

these 2 data sets was 47 Da. This result further confirms that no allelic ladders 

or other internal DNA standards are needed to obtain accurate measurements 

with mass spectrometry. 
60

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

Allele 6 Allele 7 Allele 8 Allele 9 Allele 10 Allele 11 Allele 12 Allele 13

Expected

mass (Da) 15,345 16,605 17,865 19,125 20,385 21,644 22,904 24,164

1 15,346 16,623 17,903 19,130 20,388 21,623 22,860 24,074

2 15,387 16,667 17,901 19,129 20,387 21,639 22,893 24,114

3 15,372 16,629 17,887 19,143 20,400 21,615 22,877 24,091

4 15,385 16,653 17,903 19,163 20,384 21,642 22,903 24,076

5 15,388 16,642 17,898 19,155 20,337 21,654 22,870 24,111

6 15,336 16,600 17,857 19,105 20,362 21,599 22,832 24,064

7 15,388 16,637 17,894 19,131 20,383 21,635 22,904 24,110

8 15,363 16,618 17,872 19,129 20,368 21,604 22,853 24,087

9 15,365 16,628 17,891 19,150 20,385 21,620 22,892 24,087

10 15,373 16,638 17,892 19,136 20,394 21,631 22,878 24,085

11 15,383 16,640 17,896 19,152 20,387 21,621 22,884 24,129

12 15,388 16,648 17,912 19,172 20,388 21,623 22,850 24,149

13 15,407 16,660 17,941 19,208 20,425 21,674 22,944 24,148

14 15,410 16,659 17,930 19,174 20,425 21,666 22,893 24,132

15 15,390 16,648 17,915 19,157 20,423 21,636 22,897 24,126

Average

mass 15,379 16,639 17,899 19,149 20,389 21,632 22,882 24,106

Std. dev. 20.2 17.8 20.6 24.7 23.7 21.0 27.2 27.3

%RSD 0.13 0.11 0.12 0.13 0.12 0.10 0.12 0.11

% error 0.22 0.21 0.19 0.13 0.02 -0.06 -0.10 -0.24

Obs-exp 33.7 34.3 34.5 23.9 4.1 -11.9 -22.0 -58.5

Exhibit 61. Fifteen replicate analyses of a TPOX allelic ladder to measure mass precision and accuracy. The precision 

was less than 30 Da for a single standard deviation, which corresponds to less than 0.1 nucleotide. The measured mass 

accuracy from the calculated expected allele masses averaged ~30 Da. Across the 8 alleles in the ladder, 120 data points are 

used to make this determination. All numbers are in Daltons (Da). Percentage error was calculated as (observed-expected)/ 

expected. This same data is also presented in histogram format; see figure 1 in Butler et al., 1998.

Sample

number

Upper Strand Expected (Da) Observed (Da)

Allele 5–6 1,211 1,210

Allele 6–7 1,211 1,211

Allele 7–8 1,211 1,215

Allele 8–9 1,211 1,215

Allele 9–9.3 907 915

Allele 9.3–10 304 306

Allele 9–10 1,211 1,221

Repeat = TCAT = 1,210.8 Da Repeat = AATG = 1,259.8 Da

= --CAT = 906.6 Da = --ATG = 946.6 Da 

Exhibit 62. Upper strand (TCAT repeat) and lower strand (AATG repeat) mass differences for the TH01 allelic ladder.

The upper strand was discernible from the lower strand due to the different sequence contents of the repeats. The STR repeat 

structure and nucleotide content can be seen using mass spectrometry. Note: The upper strand mass difference between alleles 

9.3 and 10 is 306 Da, or a “T,” and the lower strand mass difference between these same two alleles is 315 Da, or an “A.” For 

more details, see Butler et al., 1998b. 

Allele 5–6 1,260 1,259

Allele 6–7 1,260 1,262

Allele 7–8 1,260 1,269

Allele 8–9 1,260 1,267

Allele 9–9.3 947 948

Allele 9.3–10 313 315

Allele 9–10 1,260 1,263

Upper Strand Expected (Da) Observed (Da)
61

RESULTS AND DISCUSSION OF STR ANALYSIS BY MASS SPECTROMETRY

D81179

0

2

4

6

8

10

12

14

16

18

Allele call (repeat #)

ABI 310

Mass Spec

D16S539

0

2

4

6

8

10

12

14

0 20 40 60 80 100 120 140 160 180

Number of Alleles

Allele calls (repeat #)

CSF1PO

0

2

4

6

8

10

12

14

Allele call (repeat #)

Exhibit 63. Comparison of ABI 310 and mass spectrometry allele calls for 90 CEPH/diversity samples. Out of 1,080 

possible allele calls with these 3 STR loci, there were 100 with no data collected (indicated as a “0” on the allele call 

axis), and only 12 calls differed between the two methods, or ~98% correlation.

Number of Alleles

Number of Alleles

0 20 40 60 80 100 120 40 160 180

0 20 40 60 80 100 120 40 160 180

ABI 310

Mass Spec

ABI 310

Mass Spec 
63

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

Work began on the development of multiplexed SNPassays in the summer of

1998 after notice that a second NIJ

grant, Development of Multiplexed

Single Nucleotide Polymorphism

Assays from Mitochondrial and YChromosome DNA for Human Identity

Testing Using Time-of-Flight Mass

Spectrometry, had been funded.

Excellent progress was made toward

the milestones on this grant, but the

work not finished because this grant

was prematurely terminated on the

part of GeneTrace in the spring of

1999. The completed work focused 

on two areas: the development of a

10-plex SNP assay from the mtDNA

control region using a single amplicon

and the development of a multiplex

PCR assay from Y-chromosome SNP

markers that involved as many as 18

loci amplified simultaneously. This

section describes the design aspects 

of multiplex PCR and SNP assays

along with the progress made toward

the goal of producing assays that

would be useful for high-throughput

screening of mitochondrial and Ychromosome SNP markers.

The approach to SNP determination

described here has essentially three

steps: (1) PCR amplification, (2)

phosphatase digestion, and (3) SNP

primer extension. Either strand of

DNA may be probed simultaneously

in this SNP primer extension assay.

PCR primers are designed to generate

an amplicon that includes one or

more SNP sites. The initial PCR reaction is performed with standard (unlabeled) primers. A phosphatase is then

added following PCR to remove all

remaining dNTPs so that they will not

interfere with the single base extension reaction involving ddNTPs.

These reactions can all be performed

in the same tube or well in a sample

tray. A portion of the phosphatasetreated PCR product is then used for

the primer extension assay. 

In the SNP primer extension assay, a

special primer containing a biotin moiety at the 5’-end permits solid-phase

capture for sample purification prior

to mass spectrometry analysis. This

primer hybridizes upstream of the SNP

site with the 3’-end immediately adjacent to the SNP polymorphic site. A

cleavable nucleotide near the 3’-end

allows the 3’-end of the primer to be

released from the immobilized portion

and reduces the overall mass of the

measured DNA molecule (exhibit 21)

(Li et al., 1999). The complementary

nucleotide(s) to the nucleotide(s) present at the SNP site is inserted during

the extension reaction. In the case of a

heterozygote, two extension products

result. Only a single base is added to

the primer during this process because

only ddNTPs are used and the dNTPs

left over from PCR are hydrolyzed with

the phosphatase digestion step. If the

extension reaction is not driven to

completion (where the primer would

be totally consumed), then both primer

and extension product (i.e., primer

plus single nucleotide) are present

after the primer extension reaction.

The mass difference between these two

DNA oligomers is used to determine

the nucleotide present at the SNP site.

In the primer extension SNP assay, the

primer acts as an internal standard and

helps make the measurement more

precise. A histogram of mass difference

measurements across 200 samples (50

per nucleotide) is shown in exhibit 64.

The ddT and ddA differ by only 9 Da

and are the most difficult to resolve as

heterozygotes or distinguish from one

Exhibit 64. Histogram of mass difference measurements for 200 samples 

(50 for each ddN). Expected masses for the dideoxynucleotides are 273.2 Da 

for ddC, 288.2 Da for ddT, 297.2 Da for ddA, and 313.2 Da for ddG. The 

overall mass precision with this set of samples was less than 2 Da. For more 

details, see Li et al., 1999. Number of observations

40

30

40

30

0

270 280 290 300 310 320

Mass (Da) 

ddC

273.2 Da

ddT

288.2 Da

ddA

297.2 Da

313.2 Da

ddG
64

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

another in terms of mass. As reported

in a recently published paper (Li et al.,

1999), this approach has been used to

reliably determine all four possible

SNP homozygotes and all six possible

heterozygotes.

Mitochondrial 

DNA Work

The control region of mtDNA, commonly referred to as the D-loop, is

highly polymorphic and contains a

number of possible SNP sites for

analysis. MITOMAP, an internet database containing fairly comprehensive

information on mtDNA, lists 408 polymorphisms over 1,121 nucleotides of

the control region (positions 16020–

576) that have been reported in literature (MITOMAP, 1999). However,

many of these polymorphisms are rare

and population specific. The present

study focused on marker sets from a

few dozen well-studied potential SNP

sites. Special Agent Mark Wilson from

the FBI Laboratory in Washington,

D.C., who has been analyzing mtDNA

for more than 7 years, recommended a

set of 27 SNPs that would give a reasonable degree of discrimination and

make the assay about half as informative as full sequencing. His recommended mtDNA sites were positions

16069, 16114, 16126, 16129, 16189,

16223, 16224, 16278, 16290, 16294,

16296, 16304, 16309, 16311, 16319,

16362, 73, 146, 150, 152, 182, 185,

189, 195, 198, 247, and 309. The

underlined sites are those reported in 

a minisequencing assay developed by

the Forensic Science Service (Tully et

al., 1996). GeneTrace’s multiplex SNP

typing efforts began with 10 of the

SNPs used in the FSS minisequencing

assay, since those primer sequences

had already been reported and studied

together.

L15997

H00401

1021 bp PCR Product

H16069 G or A

H16129 C or T

H16189 A or G

H16224 A or G

H16311 A or G

H00073 T or C

L00146 T or C

H00152 A or G

L00195 T or C

H00247 C or T

HV1 HV2

* * * * * * * *

 * *

Exhibit 65. Schematic representation of the mtDNA control region 10-plex 

SNP assay. The asterisks represent the relative positions of the SNP sites and 

the strand that is probed.

Exhibit 66. SNP ions impacting multiplex design. Doubly charged and triply 

charged ions from higher mass primers can interfere with singly charged ions 

of smaller mass primers if the multiplex is not well designed. 

2,000 3,000 4,000 5,000 6,000 7,000

Extension

product

SNP primer

Primer

impurity

2+ ions

3+ ions

1+ ions

Na adduct

Na adduct

m/z

Signal
65

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

The reported FSS sequences were

modified slightly by removing the

poly(T) tail and converting the degenerate bases into the most common

sequence variant (identified by examination of MITOMAP information at

the appropriate mtDNA position). A

cleavable base was also incorporated at

varying positions in different primers

so that the cleaved primers would be

resolvable on a mass scale. Exhibit 26

lists the final primer set chosen for a

10-plex SNP reaction. Eight of the

primers detected SNPs on the “heavy”

GC-rich strand and two of them identified SNPs on the “light” AT-rich

strand of the mtDNA control region

(exhibit 65). Five of the primers

annealed within hypervariable region I

(HV1) and five annealed within hypervariable region II (HV2). All of the 10

chosen SNP sites were transitions of

either A to G (purine-to-purine) or

C to T (pyrimidine-to-pyrimidine)

rather than transversions (purine-topyrimidine).

Besides primer compatibility (i.e., lack

of primer dimer formation or hairpins), another important aspect of

multiplex SNP primer design is the

avoidance of multiple-charged ions.

Doubly charged and triply charged

ions of larger mass primers can fall

within the mass-to-charge range of

smaller primers. Depending on the

laser energy used and matrix crystallization, the multiple-charged ions can

be significantly abundant (exhibit 66).

Primer impurities, such as n-1 failure

products, can also impact how close

together primers can be squeezed on

a mass scale. These primer synthesis

failure products will be ~300 Da smaller in mass than the full-length primer.

Since an extension product ranges

from 273 (ddC) to 313 Da (ddG) larger than the primer itself, a minimum

of 650–700 Da is needed between

adjacent primers (postcleavage mass)

if primer synthesis failures exist to

avoid any confusion in making the

correct SNP genotype call.

Primer synthesis failure products were

observed to become more prevalent

for larger mass primers. Because resolution and sensitivity in the mass spectrometer decrease at higher masses,

it is advantageous to keep the multiplexed primers in a fairly narrow mass

window and as small as possible. The

primers in this study ranged from

1,580 to 6,500 Da. Exhibit 23 displays

the expected primer masses for the

mtDNA SNP 10-plex along with their

doubly and triply charged ions. The

smallest four primers, in the mass

range of 1,580–3,179 Da, had primer

and extension masses that were similar to multiple-charged ions of larger

primers. For example, in the bottom

panel of exhibit 6, which shows the

10-plex primers, the doubly charged

ion from MT4e (3,250 Da), which

probes site L00195, fell very close

to the singly charged ion from MT3’

(3,179 Da), which probes site H16189.

The impact of primer impurity products can also be seen in exhibit 6. An

examination of the extension product

region from primer MT7/H00073

(~6,200 Da) shows two peaks where

only one was expected (top panel).

The lower mass peak in the doublet

is labeled as “+ddC” (6,192 Da), but

the larger peak in the doublet was a

primer impurity of MT4e/L00195

(6,232 Da). The mass difference

between these two peaks was 40 Da

or exactly what one would expect for

a C/G heterozygote extension of primer

MT7/H00073. Thus, to avoid a false

positive, it was important to run the

10 primers alone as a negative control

to verify any primer impurities.

To aid development of this multiplex

SNP assay, large quantities of PCR

product were produced from K562

genomic DNA (enough for ~320 reactions) and were pooled together so

that multiple experiments would have

the same starting material. With the

K562 amplicon pool, the impact of

primer concentration variation was

examined without worrying about the

DNA template as a variable. The K562

amplicon pool was generated using

the PCR primers noted in exhibit 26,

which produced a 1,021 bp PCR product that spanned the entire D-loop

region (Wilson et al., 1995). Thus, all

10 SNP sites could be examined from

a single DNA template.

Using ABI’s standard sequencing procedures and dRhodamine dye-terminator

sequencing kit, this PCR pool was

sequenced to verify the identity of the

nucleotide at each SNP site in the 10-

plex. The sequencing primers were

the same as those reported previously

(Wilson et al., 1995). Identical results

were obtained between the sequencing

and the mass spectrometer, which verified the method (exhibit 6).

A variety of primer combinations and

primer concentrations were tested on

the way to obtaining results with the

10-plex. For example, a 4-plex and 

a 6-plex were developed first with

primers that were further apart in

terms of mass and, therefore, could be

more easily distinguished. An early 6-

plex was published in Electrophoresis

(Li et al., 1999). Primer concentrations were balanced empirically by

first running all primers at 10 pmol

and then raising or lowering the

amount of primer in the next set to

obtain a good balance between those

in the multiplex primer mix. In general, a higher amount of primer was

required for primers of higher mass.

However, this trend did not always

hold true, probably because ionization

efficiencies in MALDI mass spectrometry differ depending on DNA sequence

content. The primer concentrations in

the final “optimized” 10-plex ranged

from 10 pmol for MT3’ (3,179 Da) to

35 pmol for MT7 (5,891 Da). Primer

extension efficiencies also varied
66

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

between primers, making optimization

of these multiplexes rather challenging.

Originally, this study set out to examine 100 samples, but due to the early

termination of the project, researchers

were unable to run this multiplex SNP

assay across a panel of samples to verify that it worked with more than one

sample. Future work could include

examination of a set of population samples and correlation to DNA sequencing results. Examination of the impact

of SNPs that are close to the one being

tested and that might impact primer

annealing also needs to be done. In

addition, more SNP sites can be developed and the multiplex could be

expanded to include a larger number

of loci.

Y-Chromosome Work

While the mtDNA work produced an

opportunity to examine the mass spectrometry factors in developing an SNP

multiplex, this work involved only a

single DNA template with multiple

SNP probes. A more common situation for multiplex SNP development

is multiple DNA templates with one

or more SNP per template. SNP sites

may not be closely spaced along the

genome and could require unique

primer pairs to amplify each section

of DNA. To test this multiplex SNP

situation, researchers investigated multiple SNPs scattered across the Y chromosome. Through a collaboration with

Dr. Oefner and Dr. Underhill, 20 Ychromosome SNP markers were examined in this study. Dr. Oefner and Dr.

Underhill have identified almost 150

SNP loci on the Y chromosome, some

of which have been reported in the literature (Underhill et al., 1997). By

examining an initial set of 20 Y SNPs

and adding additional markers as needed, researchers attempted to develop

a final multiplex set based on ~50 Y

SNP loci. The collaboration provided

detailed sequence information on bases

around the SNP sites (typically several

hundred bases on either side of the

SNP site), which is important for multiplex PCR primer design. Dr. Oefner

also provided a set of 38 male genomic

DNA samples from various populations

around the world for testing purposes. 

The sequences were provided in two

batches of 10 sequences each. In the

first two sets, primer designs were

attempted for a 9-plex PCR and a 17-

plex PCR, respectively. Due to primer

incompatibilities, it was impossible to

incorporate all SNPs into each multiplex set. However, with a larger set of

sequences to choose from, it is conceivable that much larger PCR multiplexes can be developed. According to

Dr. Underhill’s nomenclature, the first

set of Y SNP markers included the following loci: M9 (C→G), M17 (1 bp

deletion, 4Gs→3Gs), M35 (G→C),

M42 (A→T), M45 (G–>A), M89

(C→T), M96 (G→C), M122 (T→C),

M130 (C→T), and M145 (G→A).

The second set of Y SNP markers contained these loci: M119 (A→C), M60

(1 bp insertion, a “T”), M55 (T→C),

PCR Mix: 5 mM MgCl2, 1X PCR buffer II, 250 μM dNTPs, 2 U TaqGold, 40 pmol 

univ-F primer, 40 pmol univ-R primer, and 0.4 pmole each locus-specific primer pair 

in 20 μL volume 

Thermal Cycling: 95 o

C for 10 min; 50 cycles at 94 oC for 30 sec, 55 oC for 30 sec, 

68 o

C for 60 sec, 72 oC for 5 min, and 4 oC forever (~3 hr total) 

Exhibit 67. Multiplex PCR information for 17-plex PCR reaction

Locus

Name

Expected

SNP

Primers

Used

Primer

Amounts

PCR Product

Size (bp)

M3 C/T F1u/R1u 0.4 pmole each 148 bp 

M17 1 bp del F1u/R1u 0.4 pmole each 149 bp

M13 G/C F1u/R1u 0.4 pmole each 172 bp 

M119 A/C F1u/R1u 0.4 pmole each 188 bp 

M2 A/G F3u/R3u 0.4 pmole each 194 bp 

M96 G/C F1u/R1u 0.4 pmole each 198 bp 

M122 T/C F2u/R2u 0.4 pmole each 208 bp 

M145 G/A F1u/R1u 0.4 pmole each 218 bp 

M45 G/A F2u/R2u 0.4 pmole each 230 bp 

M35 G/C F1u/R5u 0.4 pmole each 233 bp 

M9 C/G F1u/R1u 0.4 pmole each 243 bp 

M55 T/C F1u/R1u 0.4 pmole each 247 bp 

M60 1 bp ins F1u/R1u 0.4 pmole each 263 bp 

M89 C/T F4u/R4u 0.4 pmole each 275 bp 

M42 A/T F1u/R1u 0.4 pmole each 300 bp 

M67 A/T F1u/R1u 0.4 pmole each 314 bp 

M69 T/C F1u/R1u 0.4 pmole each 326 bp 

M26 G/C F1u/R1u 0.4 pmole each 333 bp 

M130 C/T F1u/R1u Included in 9-plex but

not 17-/18-plex PCR

155 bp

M20 primers are not compatible with the other loci in the multiplex.
67

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

M20 (A→G), M69 (T→C), M67

(A→T), M3 (C→T), M13 (G→C), 

M2 (A→G), and M26 (G→A).

Multiplex PCR primers were designed

with a UNIX version of Primer 3

(release 0.6) (Rozen et al., 1998) that

was adapted at GeneTrace by Nathan

Hunt to utilize a mispriming library

and Perl scripts for input and export

of SNP sequences and primer information, respectively. The PCR primer

sequences produced by Hunt’s program are listed in exhibit 27. The 

universal tags attached to each primer

sequence aid in multiplex compatibility

(Shuber et al., 1995). This tag added

23 bases to the 5’-end of the forward

primers and 24 bases to the 5’-end of

the reverse primers and, therefore,

increased the overall length of PCR

products by 47 bp. The addition of

the universal tag makes multiplex PCR

development much easier and reduces

the need to empirically adjust primer

concentrations to balance PCR product quantities obtained from multiple

loci (Ross et al., 1998).

To compare the amplicon yields from

various loci amplified in the multiplex

PCR, the product sizes were selected

to make them resolvable by CE separation. Thus, the PCR product sizes

ranged from 148 bp to 333 bp (exhibit

67) using the primers listed in exhibit

27. To make sure that each primer pair

worked, each marker was amplified

individually as well as in the multiplex

set using the same concentration of

PCR primers. A substantial amount of

primers remaining after PCR indicated

that the PCR efficiency was lower for

that particular marker (exhibit 68).

Researchers were able to demonstrate

male-specific PCR with a 17-plex set

of PCR primers. The male test sample

AM209 from an Amish CEPH family

(exhibit 25) produced amplicons for

17 Y SNP loci, while K562 genomic

DNA yielded no detectable PCR product because it is female DNA and,

therefore, does not contain a Y chromosome (exhibit 7).

SNP primers were designed and synthesized for probing the SNP sites

either in a singleplex (exhibit 69) or

a multiplex (exhibit 70) format. Some

additional SNP primers and multiplex

PCR primers were also designed for

testing 12 autosomal SNPs throughout

the human genome with the hope of

comparing the informativeness of

SNPs to STRs (exhibit 71). Analysis 

of these same 12 SNPs was recently

demonstrated in a multiplex PCR and

SNP assay by PerSeptive Biosystems

(Ross et al., 1998).

Optimal SNP markers for identity

testing typically have allele frequencies of 30–70% in a particular human

population. By way of comparison,

highly polymorphic STRs can have

10–15 or more alleles with allele frequencies below 15% (i.e., more alleles

and lower allele frequencies). The

characteristics of STR and SNP markers are compared in exhibit 72. SNPs

have the capability of being multiplexed to a much higher level than

STRs; however, more SNP markers are

required for the same level of discrimination compared with STRs. Only time

will tell what role new SNP markers

will have in human identity testing.

17-plex PCR

(34 primers)

Primer

dimer

Remaining

primers 233 bp

M35

314 bp

M67

326 bp

M69

333 bp

M26

1,2

3

8

12 15

 4

5

 6

7

9

10

11

13

14

16 17

10

15

16

17

Exhibit 68. CE electropherograms showing a multiplex PCR sample 

compared with individual PCR reactions. The M67 primers are more 

efficient as fewer primers remain following the PCR reaction.

7.5 10.0

Minutes
68

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

Primer Name Primer Sequence (5’→3’) Cleavage Mass (Da) Expected SNP

M145-P1 b-CTTGCCTCCACGAC(T)TTCCT 1,491 A/G

M35-P1 b-CGGAGTCTCTGCC(T)GTGTC 1,556 C/G

M9-P1 b-AACGGCCTAAGATGG(T)TGAAT 1,564 C/G

M26-P1 b-AGGCCATTCAGTG(T)TCTCTG 1,820 C/G

M67-P1 b-TTGTTCGTGGACCCC(T)CTATAT (overlaps PCR reverse primer) 1,828 A/T

M45-P1 b-CCTCAGAAGGAGC(T)TTTTGC 1,835 C/T

M145-P2 b-GATTAGGCTAAGGC(T)GGCTCT 1,845 C/T

M119-P1 b-TTCCAATTCAGCA(T)ACAGGC 1,863 T/G

M55-P1 b-GCCCCTGGATGGTT(T)AAGTTA 1,877 C/T

M20-P1 b-ACCAACTGTGGAT(T)GAAAAT (no PCR primers designed) 1,886 A/G

M122-P1 b-TCAGATTTTCCCC(T)GAGAGC 1,903 A/G

M96-P1 b-TTGGAAAACAGGTCTC(T)CATAATA 2,150 C/G

M69-P1 b-GAGGCTGTTTACAC(T)CCTGAAA 2,151 C/T

M130-P1 b-GGGCAATAAACCT(T)GGATTTC (overlaps PCR forward primer) 2,173 C/T

M42-P1 b-CACCAGCTCTCTTTTTCAT(T)ATGTAGT 2,198 A/T

M89-P1 b-CAACTCAGGCAAAG(T)GAGAGAT (overlaps PCR reverse primer) 2,232 A/G

M3-P1 b-GGGTCACCTC(T)GGGACTGA 2,537 A/G

M2-P1 b-CCTTTATCC(T)CCACAGATCTCA (overlaps PCR reverse primer) 3,636 C/T

M13 Not designed C/G

M60 Not designed (1bp insertion) ins

M17 Not designed (1bp deletion) del

Exhibit 69. Primers for testing Y-chromosome SNP markers in singleplexes. Primers were designed automatically with 

UNIX SNP probe scripts written by Nathan Hunt but have not yet been tested.

Primer Name Primer Sequence (5’→3’) Cleaved Mass (Da) Expected SNP

Y1 (M42b-a) b-CCAGCTCTCTTTTTCATTA(T)GTAGT 1,580 T/A

Y2 (M96t-b) b-CTTGGAAAACAGGTCTC(T)CATAATA 2,150 C/G

Y3 (M35b-c) b-TTCGGAGTCTC(T)GCCTGTGTC 2,768 C/G

Y4 (M130b-b) b-CCTT(T)CCCCTGGGCAG 3,380 C/T

Y5 (M145b-b) b-GATTAGGC(T)AAGGCTGGCTCT 3,723 A/G

Y6 (M122t-c) b-TAGAAAAGCAAT(T)GAGATACTAATTCA 4,333 C/T

Y7 (M45t-d) b-AAATTGGCAG(T)GAAAAATTATAGATA 4,694 A/G

Y8 (M9b-c) b-ACATGTCTAAA(T)TAAAGAAAAATAAAGAG 5,354 C/G

Y9 (M89t-e) b-CTTCC(T)AAGGTTATGTACAAAAATCT 6,210 C/T

Exhibit 70. Y SNP multiplex primer information. Primers were designed manually for multiplex SNP assay (9-plex) 

with nonoverlapping masses. 
69

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

SNP markers are not designed for multiplexing.

SNP Primer

Name Primer Sequence (5’→3’) 

Cleavage

Mass (Da)

Expected

(SNP)

C6-P1 b-GGGGACAGCCA(T)GCACTG 1,854 A/C

A2M-P1 b-GAAACACAGCAGCTTAC(T)CCAGAG 1,863 A/G

LDLR-P1 b-CCTATGACACCGTCA(T)CAGCAG 1,863 A/G

IL1A-P4 b-TTTTAGAAATCATCAAGCC(T)AGGTCA 1,878 T/G

CD18-P2 b-GGACATAGTGACCG(T)GCAGGT 1,894 C/T

IGF2-P1 b-CCACCTGTGATT(T)CTGGGG 1,910 C/T

ALDOB-P1 b-CGGGCCAAGAAGG(T)ATCTACC 2,102 A/G

PROS1-P1 b-CATAATGATATTAGAGCTCAC(T)CATGTCC 2,118 A/G

NF1-P1 b-CGATGGTTGTATTTGTCACCA(T)ATTAATT 2156 A/G

AT3-P0 b-ATCTCCA(T)GGGCCCAGC 2,787 C/T

CYP2D6-P1 b-GCAGCTTCAATGA(T)GAGAACCTG 2,810 C/T

LIPC-P0 b-AACATGGCT(T)CGAGAGAGTTG 3,483 A/C

Primer

Name Primer Sequence (5’→3’)

PCR

Product Size

AT3-F1u ATT TAG GTG ACA CTA TAG AAT ACT GAG ACC TCA GTT TCC TCT TCT G

AT3-R1u TAA TAC GAC TCA CTA TAG GGA GAC CCT GGT CCC ATC TCC TCT AC

159 bp

C6-F1u ATT TAG GTG ACA CTA TAG AAT ACA TCT GTC TTG CGT CCC AGT C

C6-R1u TAA TAC GAC TCA CTA TAG GGA GAC TCT TGC AGT CAG CCT CTT CA

160 bp

IGF2-F1u ATT TAG GTG ACA CTA TAG AAT ACA GTC CCT GAA CCA GCA AAG A

IGF2-R1u TAA TAC GAC TCA CTA TAG GGA GAC TTT TCG GAT GGC CAG TTT AC

163 bp

LIPC-F1u ATT TAG GTG ACA CTA TAG AAT ACA ACA CAC TGG ACC GCA AAA G

LIPC-R1u TAA TAC GAC TCA CTA TAG GGA GAC ACC CAG GCT GTA CCC AAT TA

173 bp

NF1-F1u ATT TAG GTG ACA CTA TAG AAT ACA AGG AGC AAA CGA TGG TTG TA

NF1-R1u TAA TAC GAC TCA CTA TAG GGA GAC TAG GTG GCT GCA AGG TAT CC

181 bp

LDLR-F5u ATT TAG GTG ACA CTA TAG AAT ACC CAC GGC GTC TCT TCC TAT

LDLR-R5u TAA TAC GAC TCA CTA TAG GGA GAC TGG TAT CCG CAA CAG AGA CA

181 bp

CYP2D6-F1u ATT TAG GTG ACA CTA TAG AAT ACG GTG CAG AAT TGG AGG TCA T

CYP2D6-R1u TAA TAC GAC TCA CTA TAG GGA GAC AGA ACA GGT CAG CCA CCA CTA

182 bp

CD18-F2u ATT TAG GTG ACA CTA TAG AAT ACA TCC AGG AGC AGT CGT TTG T

CD18-R2u TAA TAC GAC TCA CTA TAG GGA GAC ATG CCG CAC TCC AAG AAG

193 bp

ALDOB-F1u ATT TAG GTG ACA CTA TAG AAT ACC ACA TTT GGG GCT TGA CTT T

ALDOB-R1u TAA TAC GAC TCA CTA TAG GGA GAC TCC TTC AGT CTC CTG TCA TCA A

231 bp

A2M-F1u ATT TAG GTG ACA CTA TAG AAT ACC TCT GCC ATG CAA AAC ACA C

A2M-R1u TAA TAC GAC TCA CTA TAG GGA GAC AAC ATT CAA GTT TCC CTT ACT CAA

247 bp

PROS1-F1u ATT TAG GTG ACA CTA TAG AAT ACT AAT GGC TGC ATG GAA GTG A

PROS1-R1u TAA TAC GAC TCA CTA TAG GGA GAC CCA GGA AAG GAC CAC AAA AT

292 bp

IL1A-F1u ATT TAG GTG ACA CTA TAG AAT ACT TTG CTT CCT CAT CTG GAT TG

IL1A-R1u TAA TAC GAC TCA CTA TAG GGA GAC AGC AGC CGT GAG GTA CTG AT

324 bp

Exhibit 71. Human autosomal SNP markers designed for testing NIH diversity panel

Multiplex PCR primers for 12-plex PCR (not all amplicons are resolvable by CE). Universal primer sequences are in color.
70

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

Mass measurement of PCR-amplified 

allele(s)

Mass spectrometry

measurement

Characteristics Short Tandem Repeats (STRs)

Occurrence in human genome ~1 in every 15 kb

General informativeness High

Marker type Di-, tri-, tetranucleotide repeat markers

Number of alleles per marker Typically >5

Current detection methods Gel/capillary electrophoresis

Multiplex capability (fluorescence) >10 markers with multiple spectral channels

Heterozygote resolution

(fluorescence)

Mobility differences between alleles

Mass difference between primer and 

extension product

Single Nucleotide Polymorphisisms (SNPs)

~1 in every 1 kb

Low (20–30% as informative as STRs)

Biallelic markers

Typically 2

Microchip hybridization

Potentially thousands on microchip

Spectral differences between labeled 

nucleotides

Exhibit 72. Characteristics of STR and SNP markers. SNPs are more common in the human genome than STRs but are 

not as polymorphic. 

STR locus K562 AM209 UP006

TH01 9.3,9.3 6,7 7,9.3

TPOX 8,9 8,9 8,8

CSF1PO 9,10 10,12 11,14

D8S1179 12,12 11,13 13,13

D21S11 29,30,31 30,30 28,31

D18S51 15,16 12,12 13,13

D3S1358 16,16 15,18 15,15

VWA 16,16 18,18 16,17

FGA 21,24w 20,25 19,22

D16S539 11,12 11,12 12,13

D5S818 11,12 11,12 10,11

D13S317 8,8 14,14 11,14

D7S820 9,11 9,10 10,12

Amelogenin X,X X,Y X,Y

Exhibit 73. STR genotypes for standard DNA templates K562, AM209, and UP006 obtained using AmpF1STR®

ProfilerPlusTM and AmpF1STR® COfilerTM fluorescent STR kits. These samples were the primary controls used for 

testing of newly developed primer sets. Alleles were not always well balanced for the K562 cell line DNA due to possible 

chromosome imbalances in the original sample. For example, the STR marker D21S11 produced three balanced alleles, 

most likely because three chromosome 21s were present in the K562 genomic DNA. In addition, the allele 24 peak (“24w”

= “weak”) for the FGA locus in K562 is only one-third the height of the allele 21 peak probably because there are more 

copies of the chromosome 4 on which the allele with 21 repeats resides. K562 genomic DNA came from Promega 

Corporation and AM209 came from CEPH pedigree 884 (Amish family). UP006 is an anonymous population sample of 

European origin purchased from Bios Laboratory. 
71

RESULTS AND DISCUSSION OF MULTIPLEX SNPS

Exhibit 74. An ABI 310 CE electropherogram showing a heterozygous sample with a 15.2 microvariant at STR locus 

D3S1358. A high degree of resolution is often necessary to resolve two closely spaced alleles in which one of them is a 

microvariant. In this case, the two alleles differ by only 2 bp even though the core repeat is a tetranucleotide. In the 

comparison with STR data collected by CDOJ, this was the only sample where a different result was obtained between 

our analysis and that performed by CDOJ on the same STR markers. CDOJ reported 14,15 for this sample. 

2,000

4,000

11BSample11 11 Blue CDOJ 9708057A -B11

COFILER ...RSample18 18 Blue COfiler LADDER

12 13 14 15 16 17 18 19

15.2

15

D3S1358

95 100 105 110 115 120 125 135 140 145 130

4,000

2,000
73

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75

PUBLISHED PAPERS AND PRESENTATIONS

From 1997 to 1999, six publications resulted from the workfunded by NIJ, and at least one

more manuscript is in preparation. All

articles were published in journals or

for conference proceedings that are

accessible and frequented by forensic

DNA scientists to ensure proper dissemination of the information.

Butler, J.M., J. Li, T.A. Shaler, J.A.

Monforte, and C.H. Becker. 1998.

Reliable genotyping of short tandem

repeat loci without an allelic ladder

using time-of-flight mass spectrometry. International Journal of Legal

Medicine 112 (1): 45–49. 

Butler, J.M., J. Li, J.A. Monforte, C.H.

Becker, and S. Lee. 1998. Rapid and

automated analysis of short tandem

repeat loci using time-of-flight mass

spectrometry. In Proceedings of the

Eighth International Symposium on

Human Identification 1997. Madison,

WI: Promega Corporation, 94–101. 

Butler, J.M., K.M. Stephens, J.A.

Monforte, and C.H. Becker. 1999.

High-throughput STR analysis by

time-of-flight mass spectrometry. In

Proceedings of the Second European

Symposium on Human Identification

1998. Madison, WI: Promega

Corporation, 121–130.

Butler, J.M., and C.H. Becker. 1999.

High-throughput genotyping of

forensic STR and SNP loci using

time-of-flight mass spectrometry. In

Proceedings of the Ninth International

Symposium on Human Identification

1998. Madison, WI: Promega

Corporation, 43–51.

Li, J., J.M. Butler, Y. Tan, H. Lin, S.

Royer, L. Ohler, T.A. Shaler, J.M.

Hunter, D.J. Pollart, J.A. Monforte, and

C.H. Becker. 1999. Single nucleotide

polymorphism determination using

primer extension and time-of-flight

mass spectrometry. Electrophoresis

20:1258–1265. 

Butler, J.M. 1999. STR analysis by

time-of-flight mass spectrometry.

Profiles in DNA 2(3): 3–6.

In addition, one patent was submitted

based on work funded by NIJ. This

patent describes the PCR primer

sequences used to generate smaller

amplicons for 33 different STR loci

along with representative mass spectrometry results. The sequences for

multiple cleavable primers are also

described, although this proprietary

chemistry is the subject of U.S. Patent

5,700,642, which was issued in

December 1997. The process of multiplexing STR loci by interleaving the

alleles on a compressed mass scale is

also claimed by U.S. Patent 6,090,558

(Butler et al., 2000).

During the course of this NIJ grant,

research findings were presented to

the forensic DNA community at the

following scientific meetings:

◆ Eighth International Symposium

on Human Identification

(September 20, 1997)

◆ San Diego Conference, Nucleic

Acid Technology: The Cutting

Edge of Discovery (November 7,

1997)

◆ NIJ Research Committee (February

8, 1998)

◆ American Academy of Forensic

Sciences (February 13, 1998)

◆ Southwest Association of Forensic

Scientists DNA training workshop

(April 23, 1998)

◆ California Association of Criminalists DNA training workshop

(May 6, 1998)

◆ National Conference on the Future

of DNA (May 22, 1998)

◆ Florida DNA Training Session

(May 22, 1998)

◆ American Society of Mass

Spectrometry (June 4, 1998)

◆ Second European Symposium on

Human Identification (June 12,

1998)

◆ IBC DNA Forensics Meeting (July

31, 1998)

◆ Ninth International Symposium on

Human Identification (October 8,

1998)

◆ Fourth Annual CODIS User’s

Group Meeting (November 20,

1998)

◆ NIJ Research Committee (February

15, 1999)

In addition, the authors participated

in NIJ’s “Technology Saves Lives”

Technology Fair on Capitol Hill in

Washington, D.C., March 30–31,

1998, an event that provided excellent

exposure for NIJ to Congress. Here,

one of the DNA sample preparation

robots was demonstrated in the lobby

of the Rayburn Building. In September

1998, an 11-minute video was also

prepared to illustrate some of the

advantages of mass spectrometry for

high-throughput DNA typing.
About the National Institute of Justice

NIJ is the research and development agency of the U.S. Department of Justice and is the only Federal agency solely dedicated

to researching crime control and justice issues. NIJ provides objective, independent, nonpartisan, evidence-based knowledge

and tools to meet the challenges of crime and justice, particularly at the State and local levels. NIJ’s principal authorities are

derived from the Omnibus Crime Control and Safe Streets Act of 1968, as amended (42 U.S.C. §§ 3721–3722).

NIJ’s Mission

In partnership with others, NIJ’s mission is to prevent and reduce crime, improve law enforcement and the administration of

justice, and promote public safety. By applying the disciplines of the social and physical sciences, NIJ—

• Researches the nature and impact of crime and delinquency.

• Develops applied technologies, standards, and tools for criminal justice practitioners.

• Evaluates existing programs and responses to crime.

• Tests innovative concepts and program models in the field.

• Assists policymakers, program partners, and justice agencies.

• Disseminates knowledge to many audiences.

NIJ’s Strategic Direction and Program Areas

NIJ is committed to five challenges as part of its strategic plan: 1) rethinking justice and the processes that create just communities; 2) understanding the nexus between social conditions and crime; 3) breaking the cycle of crime by testing researchbased interventions; 4) creating the tools and technologies that meet the needs of practitioners; and 5) expanding horizons

through interdisciplinary and international perspectives. In addressing these strategic challenges, the Institute is involved in the

following program areas: crime control and prevention, drugs and crime, justice systems and offender behavior, violence and

victimization, communications and information technologies, critical incident response, investigative and forensic sciences

(including DNA), less-than-lethal technologies, officer protection, education and training technologies, testing and standards,

technology assistance to law enforcement and corrections agencies, field testing of promising programs, and international crime

control. NIJ communicates its findings through conferences and print and electronic media.

NIJ’s Structure

The NIJ Director is appointed by the President and confirmed by the Senate. The NIJ Director establishes the Institute’s objectives, guided by the priorities of the Office of Justice Programs, the U.S. Department of Justice, and the needs of the field. NIJ

actively solicits the views of criminal justice and other professionals and researchers to inform its search for the knowledge and

tools to guide policy and practice.

NIJ has three operating units. The Office of Research and Evaluation manages social science research and evaluation and crime

mapping research. The Office of Science and Technology manages technology research and development, standards development, and technology assistance to State and local law enforcement and corrections agencies. The Office of Development and

Communications manages field tests of model programs, international research, and knowledge dissemination programs. NIJ is

a component of the Office of Justice Programs, which also includes the Bureau of Justice Assistance, the Bureau of Justice

Statistics, the Office of Juvenile Justice and Delinquency Prevention, and the Office for Victims of Crime. 

To find out more about the National Institute of Justice, please contact:

National Criminal Justice Reference Service

P.O. Box 6000

Rockville, MD 20849–6000

800–851–3420

e-mail: askncjrs@ncjrs.org

To obtain an electronic version of this document, access the NIJ Web site

(http://www.ojp.usdoj.gov/nij).

If you have questions, call or e-mail NCJRS.