Title: Improved Analysis of DNA Short Tandem Repeats With Time-of-
Flight Mass Spectrometry
Series: Science and Technology Research Report
Author: John M. Butler and Christopher H. Becker
Published: National Institute of Justice, October 2001
Subject: Technology in law enforcement
54 pages
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U.S. Department of Justice
Office of Justice Programs
National Institute of Justice
Improved Analysis of DNA Short Tandem Repeats 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
World Wide Web Site
http://www.ojp.usdoj.gov
National Institute of Justice
World Wide Web Site
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
---------------------------
NIJ
Sarah V. Hart
Director, National Institute of Justice
Lois Tully
Project Monitor
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.
---------------------------
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.
---------------------------
Contents
Acknowledgments
Executive Summary
o Introduction
o Purpose of the Report
o Short Tandem Repeats
o Single Nucleotide Polymorphisms
o Conclusions and Implications
Project Description
o STR Grant
o SNP Grant
Scope and Methodology
o Assay Development and Primer Testing
o Sample Cleanup and Mass Spectrometry
o Sample Genotyping
o Comparison Tests With ABI 310 Genetic Analyzer
Results and Discussion of STR Analysis by Mass Spectrometry
o Marker Selection and Feasibility Studies With STR Loci
o Multiplex STR Work
o Comparison Tests Between ABI 310 and Mass Spectrometry Results
o PCR Issues
o Analytical Capabilities of This Mass Spectrometry Method
Results and Discussion of Multiplex SNPs
o Mitochondrial DNA Work
o Y-Chromosome Work
References
Published Papers and Presentations
---------------------------
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.
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 about 500,000 samples, more than 7 million genotypes
must be generated. Using currently available technologies, an estimated
$25 million (about $50/sample) and more than 5 years for well-trained and
well-funded 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 Plus[TM] and COfiler[TM] 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 GenePrint[TM] 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 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 5 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 laser-induced 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 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 about 100 billion as seen with the 13 CODIS STRs.
The perceived value of SNPs for DNA typing in a forensic setting include
the following:
 o More rapid analysis.
 o Cheaper costs.
 o Simpler interpretation of results because there are no stutter products.
 o 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 male-specific 17-plex PCR
of 17 different Y SNP markers (exhibit 7).
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:
 o Rapid results--STR typing at a rate of seconds per sample.
 o Accuracy--no allelic ladders.
 o Direct DNA measurement--no fluorescent or radioactive labels.
 o Automated sample preparation and data collection.
 o High-throughput capabilities of thousands of samples daily per system.
 o 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.
---------------------------
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:
 o 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).
 o Demonstrate multiplexing capabilities to a level of 2 or 3 for detection
with the TOF-MS method (exhibits 4-5 and 8-10).
 o Transfer the sample preparation protocols from manual to a highly
parallel and automated pipetting robot.
 o Develop a large data set to confirm the accuracy and reliability of this
method (exhibits 11-19).
 o 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.
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:
 o Produce and test a set of 10 or more SNP probes for mtDNA control
region "hot spots" (exhibit 5).
 o Develop software for multiplex SNP analysis and data interpretation.
 o Examine individual Y-chromosome SNP markers.
 o Develop multiplex PCR and multiplex SNP probes for Y-chromosome
SNP loci (exhibit 7).
 o Determine the discriminatory power for a set of Y-chromosome markers
by running about 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.
---------------------------
Scope and Methodology
Both the STR and the SNP genotyping assays used in this project involve
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.
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 recent-ly
with Primer 3 version 0.2 from the World Wide Web
(http://www-genome.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 in-house
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 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).
Human DNA samples used
Human genomic DNA samples representing several ethnic groups
(African-American, 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 (GenePrint[TM] 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 ladders for the following STR loci: AmpF1STR[R] Green
I (CSF1PO, TPOX, TH01, amelogenin), AmpF1STR[R] Blue (D3S1358,
VWA, FGA), AmpF1STR[R] Green II (amelogenin, D8S1179, D21S11,
D18S51), AmpF1STR[R] Yellow (D5S818, D13S317, D7S820), and
AmpF1STR[R] COfiler[TM] (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 FTA[TM] paper
(Life Technologies, Rockville, MD). Sample punches were removed from
the dried FTA[TM] paper card with a 1.2 mm Harris MICRO-
PUNCH[TM] (Life Technologies). The recommended washing protocol of
200 micro-L was reduced to 25 or 50 micro-L 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[R] Profiler[TM]
kit, which consists of AmpF1STR[R] 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[R]
COfiler[TM] 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[R] Profiler Plus[TM] and
AmpF1STR[R] COfiler[TM] 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 micro-L volumes with 20 pmol (1 micro-M) both
forward and reverse primers and a PCR reaction mix containing
everything else. The 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 micro-M
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 micro-L of PCR mix times the number of samples (+
about 10% overfill) with 0.2 micro-L AmpliTaq Gold[TM] DNA
polymerase (ABI) and the appropriate volume and quantity of forward and
reverse primers to bring them to a concentration of 1 micro-M 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 degrees C for 2 min
35 cycles:
  94 degrees C for 30 sec
  50, 55, or 60 degrees C for 30 sec
  72 degrees C for 30 sec
72 degrees C for 5 or 15 min
4 degrees C hold
The final incubation at 72 degrees C favors nontemplated nucleotide
addition (Clark 1988, Kimpton et al., 1993). This final incubation
temperature was dropped to 60 degrees C 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 degrees C for 11 min (to activate the TaqGold DNA polymerase)
40 cycles:
  94 degrees C for 30 sec
  55 degrees C for 30 sec
  72 degrees C for 30 sec
60 degrees C for 15 min
4 degrees C hold
Primers were typically designed to have an approximate annealing
temperature of 57-63 degrees C and thus worked well with a 55 degrees C
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 degrees 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 degrees C for 10 min
50 cycles:
  94 degrees C for 30 sec
  55 degrees C for 30 sec
  68 degrees C for 60 sec
72 degrees C for 5 min
4 degrees C hold
The PCR master mix contained 5 mM MgCl2, 2 U AmpliTaq Gold with
1X 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. "Drop-out"
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 micro-L 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 micro-L aliquot of the amplicon in 49 micro-L 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 micro-L
PCR reaction and then incubated at 37 degrees C for 60 minutes followed
by heating at 75 degrees C for 15 minutes. The SNP extension reaction
consisted of a 5 micro-L aliquot of the SAP-treated PCR product, 1X
TaqFS buffer, 1.2-2.4 U TaqFS (ABI), 12.5 micro-M dideoxynucleotide
triphosphate (ddNTP) mix, and 0.5 micro-M biotinylated, cleavable SNP
primer in a 20 micro-L volume. For multiplex analysis, SNP primer
concentrations were balanced empirically, typically in the range of 0.3-1.5
icro-M, 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 degrees C for 1 min and 25-35 cycles at
94 degrees C for 10 sec, 45-60 degrees C (depending on the annealing
temperature of the SNP primer) for 10 sec, and 70 degrees C for 10 sec.
An annealing temperature of 52 degrees C 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 streptavidin-coated 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[R]-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 micro-L
of matrix (manual protocol) or 2 micro-L 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, about 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 high-throughput instruments.
Automated high-throughput mass spectrometer
GeneTrace has designed and custom-built 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-to-noise level
is related to the number of laser shots collected. In general, signal-to-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 Gene-Trace that improved data quality and
involved several multipoint Savitzky-Golay 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 (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, well-resolved 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 about
8,000 Da for STRs, and a delay of 250-500 nanoseconds was used with a
signal blanking below about 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[R] 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.
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[R] 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.
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[R] Profiler Plus[TM] or AmpF1STR[R] COfiler[TM]
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 a 47-cm (50 micro-m
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
degrees C. DNA sizing was performed with ROX-labeled GS500 as the
internal sizing standard. Samples were prepared by adding 1 micro-L PCR
product to 20 micro-L deionized formamide containing the ROX-GS500
standard. The samples were heat-denatured at 95 degrees C 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 micro-L described in the
ABI protocol (AmpF1STR[R] ProfilerPlus[TM], 1998, and
AmpF1STR[R] Cofiler[TM], 1998) to 5 micro-L, which corresponded
with a cost reduction of 90% per DNA amplification. The kit reagents
were mixed in their ABI-specified proportions--11 micro-L primer mix, 1
micro-L TaqGold polymerase (5 U/micro-L), and 21 micro-L PCR mix. A
3 micro-L aliquot of this master mix was then added to each tube along
with 2 micro-L of genomic DNA template (typically at 1-2 ng/micro-L).
Both PE9700 and MJ Research thermal cyclers worked for
this reduced PCR volume method provided that the 200 micro-L 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
micro-L is needed for CE sample preparation; a sample that can be
re-injected multiple times if needed. Thus, with a 50 micro-L PCR
reaction, 49 micro-L were never used to produce a result under the
standard ABI protocol. A 5 micro-L 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 micro-L 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[R] Profiler[TM] kit on an ABI 310 Genetic Analyzer and
provided those genotypes for comparison purposes. These results provided
an independent verification of our work.
---------------------------
Results and Discussion of STR Analysis by Mass Spectrometry
In 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 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
time-of-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 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 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 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 about 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 about 12,000 Da (primarily only the repeat region). The repeat region
contained 44 nt (4 nt x 11 repeats) or about 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).
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 about
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 micro-L aliquot of extracted genomic DNA in a
96-well tray with each sample at a concentration of 1 ng/micro-L. A 5
micro-L 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[R] COfiler[TM] STR multiplex. Only 2 ng of genomic DNA
were used per reaction with the AmpF1STR[R] COfiler[TM] 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 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 5 88
samples 5 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[R] Profiler Plus[TM] 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 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 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.,
About 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 degrees C or 72 degrees C after the 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[R] GenoTYPE[TM] 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 (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 detection (exhibit 35). There are two possibilities for this
reduction:
 o 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 about 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 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.
 o 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 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' to 3' exonuclease was
added to the primer mix and heated to 37 degrees C 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 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 about 10,000 Da to about 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 full-length alleles for a tetranucleotide repeat, such
as AATG, differ in mass by 1,260 Da.
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 (about 600 Da) may be obtained for DNA molecules up to a mass of
about 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 about 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 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 Gene-Trace'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 about 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 TaqGold[TM] 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 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 an about 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.
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
time-span, revealing that masses can be remarkably similar, even when
data points are recollected months later. Exhibit 60 compares 57 allele
measurements 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. Gas-phase 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 about 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.
---------------------------
Results and Discussion of Multiplex SNPs
Work began on the development of multiplexed SNP assays in the summer
of 1998 after notice that a second NIJ grant, Development of Multiplexed
Single Nucleotide Polymorphism Assays from Mitochondrial and
Y-Chromosome 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 Y-chromosome 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 phosphatase-treated 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 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.
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-to-pyrimidine).
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 about 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 (about 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 about 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 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 Y-chromosome 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 about 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 to G), M17 (1 bp deletion, 4Gs to 3Gs), M35 (G to C), M42 (A to
T), M45 (G->A), M89 (C to T), M96 (G to C), M122 (T to C), M130 (C to
T), and M145 (G to A). The second set of Y SNP markers contained these
loci: M119 (A to C), M60 (1 bp insertion, a "T"), M55 (T to C), M20 (A to
G), M69 (T to C), M67 (A to T), M3 (C to T), M13 (G to C), M2 (A to G),
and M26 (G to 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.
---------------------------
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---------------------------
Published Papers and Presentations
From 1997 to 1999, six publications resulted from the work funded 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:
 o Eighth International Symposium on Human Identification (September
20, 1997)
 o San Diego Conference, Nucleic Acid Technology: The Cutting Edge of
Discovery (November 7, 1997)
 o NIJ Research Committee (February 8, 1998)
 o American Academy of Forensic Sciences (February 13, 1998)
 o Southwest Association of Forensic Scientists DNA training workshop
(April 23, 1998)
 o California Association of Criminalists DNA training workshop (May 6,
1998)
 o National Conference on the Future of DNA (May 22, 1998)
 o Florida DNA Training Session (May 22, 1998)
 o American Society of Mass Spectrometry (June 4, 1998)
 o Second European Symposium on Human Identification (June 12, 1998)
 o IBC DNA Forensics Meeting (July 31, 1998)
 o Ninth International Symposium on Human Identification (October 8,
1998)
 o Fourth Annual CODIS User's Group Meeting (November 20, 1998)
 o 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
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understanding the nexus between social conditions and crime; 3) breaking
the cycle of crime by testing research-based 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.