A Rapid and Inexpensive 

PCR-Based STR Genotyping 

Method for Identifying 

Forensic Specimens

Doris M. Kupfer1

Mark Huggins2

Brandt Cassidy3

Nicole Vu3

Dennis Burian1

Dennis V. Canfield1

1

Civil Aerospace Medical Institute

P.O. Box 25082

Oklahoma City, OK 73125

2

Advancia

655 Research Parkway

Oklahoma City, OK 73104 

3

DNA Solutions

840 Research Parkway

Oklahoma City, OK 73104

June 2006

Final Report

DOT/FAA/AM-06/14

Office of Aerospace Medicine

Washington, DC 20591
NOTICE

This document is disseminated under the sponsorship 

of the U.S. Department of Transportation in the interest 

of information exchange. The United States Government 

assumes no liability for the contents thereof.

___________

This publication and all Office of Aerospace Medicine technical reports are 

available in full-text from the Civil Aerospace Medical Institute’s publications 

Web site: 

www.faa.gov/library/reports/medical/oamtechreports/index.cfm
i

Technical Report Documentation Page 

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. 

DOT/FAA/AM-06/14 

4. Title and Subtitle 5. Report Date 

A Rapid and Inexpensive PCR-Based STR Genotyping Method for June 2006 

Identifying Forensic Specimens 6. Performing Organization Code 

 

7. Author(s) 8. Performing Organization Report No. 

Doris M. Kupfer, 1 Mark Huggins,2 Brandt Cassidy,3 Nicole Vu,3 Dennis 

Burian,1

 and Dennis V. Canfield1

 

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) 

 

11. Contract or Grant No. 

1

FAA Civil Aerospace Medical Institute 

P.O. Box 25082 

Oklahoma City, OK 73125 

2

Advancia

655 Research Parkway 

Oklahoma City, OK 73104 3

DNA Solutions 

840 Research Parkway 

Oklahoma City, OK 73104 

 

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered 

Office of Aerospace Medicine 

Federal Aviation Administration 

800 Independence Ave., S.W. 

Washington, DC 20591 14. Sponsoring Agency Code 

15. Supplemental Notes 

 

16. Abstract 

Situations arise where forensic samples can be inadvertently misidentified during field collection. Samples 

identified in the field as being from the same person that return conflicting toxicological results may suggest such 

misidentification. Polymerase chain reaction (PCR)-based human identity testing provides a reliable and 

independent method to confirm sample identification. In the study presented here, the Federal Bureau of 

Investigation’s human Combined DNA Identity System (CODIS) loci were used on a rapid, inexpensive 

microfluidics chip electrophoresis platform to confirm the identity of forensic samples from an aircraft accident 

site. Absolute allele identification was not achieved with this method, but it was found to be suitable for 

comparative analysis, as demonstrated by validation of the results and conclusions from capillary electrophoresis. 

The nine CODIS Short Tandem Repeat loci and a gender discrimination locus used in this study have a greater 

than 5E+07 matching probability suitable for small sample sizes. 

 

17. Key Words 18. Distribution Statement 

 

 

Forensic Science, Aerospace Medicine, DNA Typing, Short 

Tandem Repeats, Microfluidics Chip

Document is available to the public through the 

Defense Technical Information Center, Ft. Belvior, VA 

22060; and the National Technical Information 

Service, Springfield, VA 22161 

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price 

Unclassified Unclassified 18 

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized 

iii

Acknowledgments

We thank Dr. Marco Scarpetta, Orchid Cellmark, for his comments and help with power 

of discrimination calculations.

1

A Rapid and Inexpensive PCR-Based STR Genotyping

Method for Identifying Forensic Specimens

Introduction

The possibility exists that samples recovered from a fatal 

accident site can be misidentified. Results from routine 

toxicological or other biochemical testing of samples that 

conflict with the preliminary identification suggest this 

possibility but are not definitive. Genotyping, a DNAbased analysis, which is not a standard part of sample 

evaluation, can serve as an additional and independent 

test for sample identification. Capillary electrophoresis 

is the method often used for analysis of the polymerase 

chain reaction (PCR) products generated in genotyping 

tests (1). This method requires expensive, dedicated instrumentation, software for analysis, and trained personnel 

potentially available only in specialized facilities. There 

is a need for a more accessible and inexpensive human 

identity testing method in instances where genotyping 

is necessary only as a confirmatory test and there are a 

minimal number of samples. 

Short tandem repeat (STR) loci are polymorphic 

regions found in the genome that are used as genetic 

markers for human identity testing. Typing of STR 

loci by polymerase chain reaction (PCR) is becoming a 

standard for nuclear DNA genotyping analysis. The FBI’s 

Combined DNA Index System (CODIS) has defined a 

core set of STR markers for human identity testing (2) 

which are highly discriminatory over a wide range of 

ethnicities. Robust primer sequences for these loci have 

been made publicly available (3) and are available online 

through the National Institute of Standards and Testing 

(NIST) Website, STRbase (www.cstl.nist.gov/div831/

strbase/) (4). The probability of samples from different 

subjects having identical alleles for all of the CODIS loci 

is very small, leading to the use of these loci in forensic 

evaluations (1, 5).

Results will be presented here describing a qualitative method of genotyping using the Agilent 2100 

Bioanalyzer as the microfluidics platform for separation 

of PCR products amplified from STR loci selected from 

tetrameric CODIS STR loci. The study demonstrates 

the novel use of this instrument for rapid, inexpensive 

electrophoresis and analysis of STR PCR products to 

allow discrimination among samples from aviation accidents. Accuracy and reproducibility were demonstrated 

with control samples from a variety of tissues and blood. 

The method was applied to an aviation case study where 

initial sample identification did not correlate with subsequent biochemical and toxicological evaluation. A 

paternity group sample set previously used for capillary 

electrophoresis (CE) system validation also was tested. 

Finally, the findings were validated on a CE system with 

agreement in all cases. 

Materials and Methods

Genomic DNA extraction. Tissue or blood was stored 

frozen at –200C. Blood was thawed and a 200 μl aliquot 

used for genomic DNA isolation with the QIAamp 

DNA mini kit, #51304 (Qiagen; Valencia, CA) using the 

protocol provided by the manufacturer. Tissue samples 

included kidney, lung, liver, muscle, and skin. A scalpel 

was used to cut and mince approximately 25 mg from 

the frozen tissue. The minced tissue was processed as 

directed by the manufacturer using the ChargeSwitch 

gDNA Mini Tissue kit, #CS11204 (Invitrogen; Carlsbad, 

CA). Yield was determined by A260 on a NanoDrop 

Spectrophotometer (Model ND-1000, NanoDrop Technologies; Wilmington, DE). Quality was examined on a 

Bioanalyzer DNA7500 chip (Agilent Technologies; Palo 

Alto, CA). Samples showing presence of material equal 

to or greater than 10Kbp were used for PCR.

Sample sets. The 15 control samples were from three 

individuals. Samples C1-C5 were from Subject 1, Samples 

C6-C10 from Subject 2, and Samples C11-C15 were 

from Subject 3. Each control sample set contained a 

sample from blood, liver, lung, kidney and muscle. The 

aviation case study contained five samples, labeled T1-

T5. They were from kidney, skin, kidney, muscle, and 

lung, respectively. The paternity set was made up of four 

blood samples, 1304-1307 from the College of American 

Pathologists, AABB/CAP PARF Survey B 2005.

PCR Primers and Amplification. Sequences for the 

ten primer pairs used in the study were as published (3) 

with modifications as shown at STRbase, www.cstl.nist.

gov/biotech/strbase/promega_primers.htm (4). Nine 

of these are from the 13 CODIS core STR loci (6) and 

one, amelogenin, is a sex-determination locus (5). Table 

1 lists the STR loci and primer sequences used for amplification and the expected product sizes. Primers were 

synthesized (Integrated DNA Technologies; Coralville, 

IA) and diluted as needed.
2

Table 1. Genetic loci and associated primer pair sequences used for PCR in the study (4 ). See 

Materials & Methods for PCR conditions. 

Locus* Primer A Primer B Product size 

range (bp) 

Repeat 

Structure

Amel ACC TCA TCC TGG 

GCA CCC TGG TT 

AGG CTT GAG GCC 

AAC CAT CAG 

212, 218 NA 

CSF1PO AAC CTG AGT CTG 

CCA AGG ACT AGC 

TTC CAC ACA CCA 

CTG GCC ATC TTC 

291-327 [AGAT]n 

D3S1358 ACT GCA GTC CAA 

TCT GGG T 

ATG AAA TCA ACA 

GAG GCT TGC 

111-143 TCTA[TCTG]n[T

CTA]n

D5S818 GGT GAT TTT CCT 

CCT

TGG TAT CC 

AGC CAC AGT TTA 

CAA CAT TTG TAT CT 

123-155 [AGAT]n 

D7S820 ATG TTG GTC AGG 

CTG ACT ATG 

GAT TCC ACA TTT 

ATC CTC ATT GAC 

211-251 [GATA]n 

D13S317 ACA GAA GTC TGG 

GAT GTG GAG GA 

GGC AGC CCA AAA 

AGA CAG A 

157-201 [TATC]n 

D16S539 GGG GGT CTA AGA 

GCT TGT AAA AAG 

GTT TGT GTG TGC 

ATC TGT AAG CAT 

GTA TC 

264-304 [GATA]n 

THOI ATT CAA AGG GTA 

TCT GGG CTC TGG 

GTG GGC TGA AAA 

GCT CCC GAT TAT 

171-215 [AATG]n 

TPOX ACT GGC ACA GAA 

CAG GCA CTT AGG 

GGA GGA ACT GGG 

AAC CAC ACA GGT 

TA

220-256 [AATG]n 

vWA† GCC CTA GTG GAT 

GAT AAG AAT AAT 

CAG TAT GTG 

GGA CAG ATG ATA 

AAT ACA TAG GAT 

GGA TGG 

123-181 [TCTA]n[TCTG]n[

TCTA]n

(TCCA[TCTA]n)

*Amel (Amelogenin) is a sex-determination locus, all others listed are tetrameric STR loci 

†

vWA repeat sequence in ( ) is not always present 
3

Amplification was performed using reaction conditions 

recommended in the Promega PowerPlex 2.1 system (7) 

except that the buffer was 1x Amplitaq gold reaction 

buffer, 0.1% TritonX-100, and 0.2mM each dNTP in a 

25μl final volume. Cycling was performed on a GeneAmp 

9600 thermal cycler (PerkinElmer; Wellesley, MA) using 

the recommended cycling profile for 40 cycles:

An initial 950 C 11 min, 960 C 1 min; then 940 C 

30sec, ramp 68 sec to 600 C, hold for 30 sec, ramp 50 

sec to 700 C, hold for 45 sec, for 10 cycles. Then 900 C 

30 sec, ramp 60 sec to 600 C, hold for 30 sec, ramp 50 

sec to 700 C, hold for 45 sec, for 30 cycles. Then 600 C 

for 30 min, 40 C soak.

Preliminary testing was performed to optimize the 

reactions for primer and template concentration. The 

conditions selected for the study were 1-5ng template 

DNA and 800 nM each primer for a 25μl reaction. 

For multiplexed reactions, conditions were unchanged 

except that the template concentrations were increased 

two-fold.

STR Electrophoresis and Analysis. No cleanup of the 

amplification products was performed. Detection of 

a 1μl aliquot from a 1:4 dilution in distilled water of 

each amplification product was performed on a 2100 

Bioanalyzer using Expert version B.01.02.S1136 software and DNA 500 Labchip kits (Agilent Technologies; 

Palo Alto, CA). Expert software was used to overlay 

the electropherograms and assign PCR product length. 

Product lengths were calculated by the software based 

on product migration times compared to spiked-in size 

standards. Capillary electrophoresis was performed on a 

310 Genetic Analyzer (Applied Biosystems; Foster City, 

CA). Amplification and labeling of PCR products for 

capillary electrophoresis were done using the PowerPlex 

16 System (Promega Corp.; Madison, WI).

Results and Discussion

The goal of this study was to develop a simple DNAbased protocol that would allow an independent evaluation of accident site samples in cases where toxicological 

testing results did not agree with the assigned sample 

identity. PCR is the recognized method of choice because 

it requires small amounts of starting DNA template, is 

highly reproducible, and is relatively insensitive to degradation for the amplicon sizes used in this study. An 

extensive human genotyping system has been established 

using PCR of well-characterized, short repetitive genomic 

regions with standardized primers. The PCR primers were 

selected from the well-characterized FBI human CODIS 

STR (short tandem repeats) loci. STRbase was used as the 

source of the CODIS primer sequences (www.cstl.nist.

gov/div831/strbase/) (4). An Agilent 2100 Bioanalyzer 

was used for the electrophoresis and analysis of PCR 

samples as a less expensive, convenient alternative to 

capillary electrophoresis. The Bioanalyzer uses microfluidics chips to perform electrophoresis in the presence of a 

fluorescent, intercalating dye. In the study presented here, 

this instrument was used for the separation and analysis 

of unpurified, unlabelled tetrameric STR PCR products 

from three sets of samples; first, a control panel of tissue and blood samples from three individuals; second, a 

case study where toxicological testing results conflicted 

with sample identification made at the accident site; and 

finally, a paternity sample set that had previously been 

examined by capillary electrophoresis (CE).

Nine primer sets from the CODIS STR loci set (6) and 

a gender identification locus, amelogenin (5) were selected 

for PCR to have an acceptable power of discrimination. 

Method development was performed using the control 

panel. The control panel consisted of liver, lung, kidney, 

muscle, and blood from each of three subjects. DNA was 

extracted from all 15 samples and the optimum primer 

and template concentrations were determined with the 

standard thermocycling protocol for PCR given in the 

Materials and Methods section. 

There were two primary concerns with the development of the assay: reproducibility across tissue types at 

each locus, and assay sensitivity. Efficacy of the Bioanalyzer for fragment sizing has been demonstrated (8) with 

DNA7500 chips, which have a 100-7500 basepair (bp) 

optimum range. This study showed reproducible sizing 

results for both well-to-well and day-to-day replicates 

using a single 380 bp PCR product and a commercial 

plasmid digest. The DNA 500 chips used in this study 

have a specified size range of 25-500 bp and at least a five 

basepair resolution. The CODIS loci used in the study are 

tetrameric. This defined the resolution necessary for the 

STR assay to be four basepairs. Table 1 lists the loci and 

primers selected for the study and the expected product 

size ranges. In the preliminary tests, a commercial 25 bp 

ladder containing 11 species was used, as well as control 

sample PCR products, to more carefully examine the 

reproducibility and resolution of the DNA 500 chip. The 

electropherograms from the test showed that there was 

a high level of intra-chip and inter-chip reproducibility. 

Table 2A shows that the CV (coefficient of variation) of 

the well-to-well basepair values for the 25 bp ladder were 

low, ranging from 0-0.46% over 6 replicates of 11 species/well. The size of the 50 bp species was called the least 

accurately, showing a 10% difference from the expected 

size (45 bp instead of 50 bp). However, species in the size 

range of the STR loci used in this study (approximately 

100-300bp) averaged approximately 2% larger than the 
4

Table 2. Well-to-well and inter chip comparison of basepair sizes from ladder standards or PCR products run on 2100 Bioanalyzer DNA 500 chips. A. Comparison among multiple runs with the Promega 25 bp ladder, all sizes are in basepairs (bp) showing standard deviation between wells and t-Test results between chips. 

Chip 1 Chip2 Chip1vs2 % Difference from Expected 

 Well %CV* Well %CV t-Statistic Mean Expected % 

10 11 12 10 11 12 45 45 45 0.00 45 45 45 0.00 NA 45 50 -10 74 74 74 0.00 75 75 75 0.00 NA 74.5 75 -0.7 

101 101 101 0.00 103 102 102 0.46 0.057 101.5 100 1.5 128 127 127 0.37 129 129 128 0.37 0.057 128 125 2.4 153 153 153 0.00 155 155 155 0.00 NA 154 150 2.7 178 178 178 0.00 180 180 180 0.00 NA 179 175 2.3 203 202 202 0.23 205 205 204 0.23 0.02 203.5 200 1.8 229 228 228 0.21 231 231 230 0.20 0.02 229.5 225 2 254 253 253 0.19 257 257 256 0.18 0.01 255 250 2 281 280 279 0.29 284 283 283 0.17 0.01 281.7 275 2.4 307 306 305 0.27 311 311 310 0.15 0.005 308.3 300 2.8 

* %CV coefficient of variation 
5

Table 2B. Comparison of the two amelogenin PCR products with sizes in basepairs. %CV 

is used to compare intrachip results, t-test results compare peaks 1 and 2 between chips. 

 Chip 1 Chip 2 

Well Peak1 Peak2 Peak1 Peak2 

2 205 209 206 210 

3 205 209 206 210 

4 204 209 206 210 

5 205 209 206 210 

6 205 209 207 210 

7 204 208 207 210 

8 204 208 206 210 

9 203 208 206 210 

%CV 0.24 0.22 0.22 0.00 

 t-Test (between 

 chips) 

Peak 1 Peak 2 

0.000383 0.000136 

expected sizes. The amelogenin PCR products from the 

control panel also showed a low CV of 0-0.24% for peaks 

1 and 2 (Table 2B). This indicated a good well-to-well 

correlation. Paired t-Tests were performed to compare 

data between chips and showed statistically significant 

differences for both the 25 bp ladder and the amelogenin 

PCR products (p<0.05, Tables 2A and B). 

Accuracy of the Bioanalyzer depended on the size of the 

molecule but was well within specifications for the chip. 

The 25bp ladder species were called consistently within 

2% of the expected size but were generally called larger 

than the expected size (Table 2A), except for the 50bp 

species, which was consistently called smaller. Table 2B 

shows that the amelogenin PCR products were consistently called within 4% of the expected size by the Expert 

software. These have been published as being 212 and 

218 bp, respectively (5) but migrate consistently as seven 

to nine basepairs smaller in our hands on the Bioanalyzer 

platform. The difference in migration may be because the 

PCR reaction was not purified or denatured before electrophoresis or to the presence of “ragged” single stranded 

ends on the PCR products, compared to the 25bp ladder 

species in the commercial product. This suggested that 

the Bioanalyzer was limited in the detection of absolute 

sizes of the genotyping PCR products. However, the 

high level of reproducibility of the intra-chip size calls 

also suggested that reliable comparative analysis would 

be possible and would allow an accurate determination 

of forensic sample identification. 

Reproducibility between different samples from the 

same subject was evaluated. DNA isolated from control 

samples C3 and C5 (isolated from lung and muscle) was 

subjected to PCR, and the products were analyzed on 

the Bioanalyzer in replicate wells. No significant difference in fragment lengths called by the Expert software 

was seen when the replicates were compared across tissue 

types (data not shown). This suggested that the isolation 

protocol yielded consistent template material regardless 

of starting tissue. 

Based on our sample identification need, the design of 

the assay was for rapid evaluation and relative comparison 

of PCR products, rather than absolute identification of the 

specific alleles displayed by the samples. Due to statistical 

significance of inter-chip sensitivity, all loci comparisons 

were done between samples electrophoresed on the same 

chip (Table 2). 

All 15 of the control samples were used for PCR with 

the ten primer pairs using the conditions listed in Materials and Methods and subjected to electrophoresis on the 

Bioanalyzer. The results were evaluated using the Expert 

software. The electrophoresis results, both aligned sizes 

(in basepairs) and electropherograms, were examined for 

patterns to discriminate between samples. It was noted 

that the occurrence of singlets (homozygous alleles) versus 

doublets (heterozygous alleles) was sufficient to discriminate between some samples such that the three control 

subjects (samples C1-C15) could be identified based on 

the pattern of homozygous alleles alone. This is a useful 

starting point for data analysis; however, homozygosity 

was not necessary to determine sample identities since 

differences at biallelic loci were observed.
6

The aviation case study contained five samples, T1-T5. 

PCR was performed using the established conditions with 

each of the ten primer sets, followed by electrophoresis 

on the Bioanalyzer. The results showed that T5 had homozygous alleles for the CSF1PO, D5S818, and THO1 

loci. This was sufficient to identify Sample T5 as being 

unique from T1-T4 (Table 3A). The basepair sizes assigned 

to the PCR products suggested that the remaining four 

samples could be divided into two groups. Samples T1 

and T3 fell into one group and Samples T2 and T4 fell 

into a second. Samples in each group potentially belong 

to the same individual based on the assigned basepair sizes 

for the PCR products (Table 3A). Examining overlays of 

the electropherograms confirmed that Samples T1 and T3 

displayed the same peaks for all loci tested and differed 

from T2 and T4. Samples T2 and T4 also displayed the 

same peaks for all loci tested. This confirmed the assignment to three groups. Figure 1 is an electropherogram 

overlay for the vWA locus PCR products from samples 

T1-T5, showing an example of the relative peak migration for the aviation case study samples. Figure 1A clearly 

shows the difference in migration pattern between peaks 

from samples T1, T2, and T5. Figure 1B shows electrophoretic migration patterns clearly demonstrating that 

samples T1 and T3 contained the same vWA alleles that 

differed from samples T2 and T4, which in turn, display 

the same vWA alleles. See Table 3A for the corresponding 

assigned basepair measurements and assessment. Combining the human identity testing information with the 

toxicological data suggested that the original assignment 

of samples T2, T4, and T5 to the same individual was an 

error. Sample T5 was clearly from a different individual. 

Samples T1 and T3 were blind controls from the same 

individual included to confirm the efficacy of the assay 

for quality assurance.

To further validate the assay, the aviation case study 

samples were examined by CE (Materials and Methods) 

to confirm the Bioanalyzer conclusions, and a set of four 

samples making up a paternity group (samples 1304-

1307), which had previously been examined by CE, was 

tested using the Bioanalyzer. The CE results are summarized in Table 3, along with the Bioanalyzer basepair 

assignments and analysis summary. Listed in Table 3A are 

the results of the aviation case study by sample with both 

the base pair size assigned by the Expert software (“bp” 

column) and the allele assignment made by the Genotyper 

software following capillary electrophoresis on an ABI 

310 (“al” column). Results were summarized using only 

the microfluidics chip electrophoresis data and agreed 

with the CE results at all loci except CSF1PO and Tho1. 

Alleles 11 and 12 for locus CSF1PO were not resolved 

sufficiently to be called with the Bioanalyzer software. 

Manual examination of the electropherograms showed 

a partially resolved doublet. In the studies reported here, 

this did not change the final conclusion for the samples. 

The poor resolution of these alleles is thought to be due 

to the larger sizes of the CSF1PO PCR products. Alleles 

11 and 12 are reported as 311 and 315 bp respectively. 

Heterozygous samples from all other loci used in the 

study with alleles differing by four basepairs were easily 

resolved. For Bioanalyzer genotyping, it is expected that 

choosing loci with PCR products of less than 300bp will 

have better sensitivity and accuracy. Alleles 9 and 9.3 for 

the Tho1 locus could not be resolved. Even with manual 

observation, a doublet call could not be made. There is 

only a three basepair size difference between these, 195 

and 198 bp, respectively. However, as shown in this 

study, it is unlikely that an inability to resolve these two 

alleles will affect the overall accuracy of the assay across 

a wide range of loci.

The paternity group samples were known to be a 

mother (1304), daughter (1305), and potential fathers 

(1306 and 1307). The microfluidics chip results (Table 

3B) clearly identified four individuals, and did not support 1307 as a potential father but did support paternity 

for 1306 for all loci tested. These findings agreed with 

the CE results.

The ability of the Bioanalyzer to resolve products 

from multiplexed PCR reactions was tested. The limited 

resolution of the Bioanalyzer chip and unlabeled PCR 

products prevented the high degree of multiplexing 

possible with the CE systems. However, a series of five 

duplex reactions were successfully resolved. This reduced 

reagent and microchip usage by half. CSF1PO and ThoI 

reactions could be cycled and electrophoresed together, as 

could D13S17 and TPOX, D3S1358 and amelogenin, 

D16S539 and D5S818, and finally, vWA and D7S820 

(see Material and Methods for conditions) with no loss 

of resolution and no overlap in the electrophoresed PCR 

products. Figure 2 shows an example of this duplex reaction set. Secondary products can be seen in the D5S818 

and D16S539 panel. However, the height and migration 

time clearly show these, as well as the shoulder seen in 

the TPOX peak, as artifacts. The duplexed loci were 

combined for optimum separation but are not the only 

multiplex reactions. A number of additional combinations were tested (data not shown), and there appeared 

to be no excessive secondary products or inhibition of 

amplification due to the presence of multiple sets of 

primers at the concentrations reported here. Indeed, a 

triplex of D5S818, CSF1PO, and THOI amplifies and 

separates well. 

To address the sensitivity of the assay to contaminated 

template samples that could result in a miscalled genotype, PCR products from a mixture of two templates 

were examined. Samples 1304 and 1306 were mixed 
7

Table 3. Bioanalyzer (bp) and capillary electrophoresis (al) results for samples in the Case Study and Paternity Group. The Bioanalyzer summary 

was supported by the CE results in all cases, see text. A. The aviation case study results with summary determined from the Bioanalyzer results. 

B. The paternity group results with summary and paternity determination from the Bioanalyzer results. 

Case Study Paternity Group 

T1 T2 T3 T4 T5 Summary 1304 1305 1306 1307 Summary Paternity

Locus bp* al bp al bp al bp al bp al bp al bp al bp al bp al Amel 204 X 204 X 204 X 204 X 204 X All male 206 X 205 X 205 X 205 X 04,05 female NA†

209 Y 209 Y 208 Y 208 Y 208 Y 209 Y 209 Y 06,07 male 

CSF1PO 309 10 310 10 1=3, 2=4 309 10 309 10 05, 06 unique not 07 

313 11 311 11 313 11 312 11 5 unique 

 316 11,12 318 11,12 318 12 317 11,12 

322 13 322 13 

D3S1358 131 14 132 14 131 14 1=3, 2=4 131 14 130 14 all unique not 07 

 135 15 136 15 5 unique 135 15 134 15 

140 16 139 16 138 16 138 16 143 17 143 17 144 17 143 17 

 146 18 

D5S818 1=3, 2=4 139 10 04, 06 unique ND‡

143 11 143 11 142 11 5 unique 142 11 141 11 141 11 148 12 147 12 147 12 146 12 146 12 145 12 145 12 

 150 13 150 13 

D7S820 1=3=5, 226 8 all unique ND 

229 9 229 9 228 9 2=4 

 233 10 233 10 235 10 234 10 

237 11 237 11 236 11 239 11 238 11 238 11 

 240 12 241 12  255 new 

D13S317 1=3, 2=4 181 8 180 8 04, 07 unique not 07 
8

Table 3 (continued). 

 5 unique 189 10 

192 11 192 11 191 11 196 12 195 12 195 12 195 12 195 12 196 12 197 12 197 12 197 12 

 204 14 203 14 

D16S539 1=3, 2=4 275 9 06, 07 unique not 07

 280 10 5 unique 279 10 

283 11 283 11 283 11 283 11 285 11 285 11 284 11 

 287 12  291 13 292 13 292 13 293 13 293 13 

296 14 295 14 

THO1 183 6 183 6 183 6 183 6 1=3, 2=4 183 6 183 6 183 6 06, 07 unique not 07

 186 7 186 7 5 unique  190 8 

196 9.3 196 9.3 195 9, 9.3 196 9.3 196 9.3 

TPOX 224 8 223 8 223 8 223 8 223 8 1=3, 224 8 224 8 225 8 225 8 04, 07 unique not 07

226 9 226 9 2=4=5 

 230 10 230 10 230 10 

 236 11  239 12 239 12 

vWA 144 14 143 14 1=3, 2=4 146 14 146 14 06, 07 unique not 07

 149 15 149 15 5 unique 150 15  153 16 153 16  158 17 158 17 158 17 

160 18 160 18 161 18 

 165 19  169 20 

*bp basepair from the Bioanalyzer assignment, al allele from the CE results †NA not applicable ‡ND not discriminatory 
9

Figure 1. Electropherogram overlays of locus vWA PCR products. The X-axis 

is migration time, the Y-axis is fluorescence units. 

Overlay of samples T1, T2, T5 show their unique migration patterns.

Overlay of samples T1, T3 and T2, T4 respectively, show matching migration 

patterns, suggesting T1=T3 and T2=T4.
10

Figure 2. Duplex PCR electropherograms for the ten STR loci used in the study for Control 

sample 2. PCR performed using conditions described in Materials and Methods. Peaks with 

intersecting horizontal lines were considered valid products for analysis by the Bioanalyzer 

software. The X-axis is migration time, the Y-axis is fluorescence units. 
11

in three proportions (Fig. 3A). Both the mixed and unmixed samples were subjected to PCR with D3S1358 

primers and the products subjected to electrophoresis 

on the microfluidics chip, as above. Control samples C1 

and C14 were similarly mixed and PCR was performed 

with D7S820 primers. In Figure 3 the electrophoresis 

profiles from both of these series are shown. Figure 3A 

shows the presence of additional peaks or shoulders in 

the 1304/1306 mixtures, clear evidence of more than 

one template source. However, the mixture of samples 

C1 and C14 shown in Figure 3B has a confusing profile 

that could result in a false genotype assignment in both 

the 75:25 and 50:50 C14:C1 mixtures due to the more 

intense homozygous C1 peak. The significantly lower 

left-most peak contributed by C14 and the shoulder on 

the C1 peak (also contributed by C14) would suggest 

the possibility of contaminated template. Therefore, 

care should be taken to look for greatly differing peak 

heights as indicators of sample contamination. These 

results suggest that only heavily contaminated samples 

will result in confusing results since contaminating peaks 

could not be seen in 10:90 and 90:10 mixtures of these 

samples (data not shown). Taken together, Figures 3A 

and B suggest that in the event of contaminated samples 

an ambiguous reading would likely be clearly diagnosed 

by results at other loci.

The statistical power of the assay was estimated using data from the Einum and Scarpetta study for three 

North American ethnic groups (9). The nine STR loci 

were determined to provide a combined matching 

probability of approximately 9.5E+07 to 3E+08 if all 

tetrameric alleles could be detected with the Bioanalyzer protocol. Removing the CSF1PO locus from the 

matching probability calculation naturally reduces the 

matching probability (Table 4) but the assay retains an 

acceptable level of power for small, defined populations. 

The assay was not able to discriminate between the fairly 

common 9 and 9.3 ThoI alleles (see Table 3). Binning 

of these two alleles, using the formula for power of 

discrimination (10), provided a correction for the ThoI 

locus (M. Scarpetta, personal communication) giving a 

corrected matching probability of approximately 5E+07 

to 1.9E+08 (Table 4). It has been observed that the 

ThoI 9.3 and 10 alleles that differ by one basepair are 

difficult to resolve on some platforms (11, 12). Allele 

10 is relatively rare and has not been detected to date 

in our samples sets, but a binning of alleles 9.3 and 10 

(performed as for the 9 and 9.3 allele binning) showed 

a small change in allelic frequencies from 0.8372 with 

no binning to 0.8355 for the Black population, 0.8873 

to 0.8824 for the Caucasian population, and 0.8295 

to 0.8272 for the Hispanic population. Binning these 

alleles would have only a minimal effect on the overall 

matching probability of this assay. The amelogenin 

gender discrimination locus also was included in the 

study and does provide additional power, but because it 

is not an STR locus, it was not included in the Einum 

and Scarpetta data. 

Table 4. Estimation of Matching Probability for the nine STR loci used in the 

study based on PD values from Einum and Scarpetta (9). 

 Matching Probability (1/1-PD)†

STR locus N. American Blacks 

N=8000 

N. American Caucasians 

N=8000 

N. American 

Hispanic N=1000 

CSF1PO* 12.66 8.26 8.06 

D5S818 9.71 7.14 8.26 

D7S820 12.05 16.13 13.51 

D13S317 7.35 13.33 17.86 

D16S539 14.49 11.36 12.82 

THOI‡ 9.71 11.90 8.62 

TPOX 11.63 5.32 6.76 

vWA 17.24 10.31 12.99 

Total MP 3.07E+08 9.42E+07 1.56E+08 

w/o CSF1PO 2.43E+07 1.14E+07 1.93E+07 

w/o THOI & CSF1PO 2.50E+06 9.57E+05 2.24E+06 

*Cannot discriminate all tetrameric alleles for this locus 

†

PD Power of Discrimination, see text 

‡ Includes correction for inability to discriminate alleles 9 and 9.3, see text. 
12

Figure 3. The appearance of PCR products from mixture of two sample templates in three proportions. 

A. Appearance of D3S1358 PCR products from mixed template Paternity group samples 1304 and 1306 in 

three proportions where the presence of both templates is easily detected. Peaks 1 and 3 are from Sample 

1304 and Peaks 2 and 4 are from Sample 1306, as indicated in the overlay panel.

B. Appearance of mixed samples following PCR with D7S820 using Control samples C1 and C14. The 

overlay shows the relative mobility of the peaks from each sample with peaks 1 and 2 from C14 and peak 3 

from C1. See text for discussion. 
13

A final note, the DNA1000 series II kit, which will 

replace the DNA500 kit, was tested with both the Promega 25 bp ladder and the case study samples and found 

to match the DNA500 resolution and reproducibility. 

There was a roughly 3.5% increase in assigned sizes for 

all peaks using the newer kit (data not shown). 

The protocol presented here is an inexpensive, rapid 

method using instrumentation available at a fraction 

of the cost of capillary electrophoresis instruments and 

shows great potential for labs screening small numbers of 

samples. DNA isolated from blood and tissue from control 

subjects established the feasibility of the Bioanalyzer for 

the separation of PCR products from STR loci using the 

well-established CODIS primers. Both an aviation case 

study and a paternity CE validation set were successfully 

analyzed with the protocol. Use of a microfluidics chip 

system precluded purification of PCR products or use of 

specialized equipment and reagents. This makes the assay 

more accessible to laboratories with basic molecular biology 

capabilities in instances where small numbers of samples 

are assayed. The analysis was done with the Bioanalyzer 

software, which determined a basepair size assignment 

following electrophoresis and for each PCR product generated an electropherogram that could be overlaid with other 

electropherograms for visual inspection of results due to 

the reliability of size determination and the comparison to 

a standard. While small discrepancies in size assignments 

are common, they can easily be addressed by viewing the 

electropherogram overlays of the size-separated PCR products. This analysis method worked well for both uniplex 

and multiplexed data. The study did reveal a shortcoming 

of the microfluidics chip platform to discriminate tetrameric n and n+1 CSF1PO alleles where products were 

greater than 300 bp. This suggested that this locus was less 

informative for genotyping on the Bioanalyzer, and loci 

that yield PCR products within the more sensitive range of 

the instrument will allow a determination of overall assay 

matching probability. The protocol takes advantage of the 

well-characterized CODIS primer sets so a large body of 

literature is available regarding characterization and use 

of the tetrameric STR loci for genotyping (for example 

4, 10). The application utilizes a relative comparison of 

electrophoretically separated products to return results 

that are accurate within the limitation of the instrumentation. Absolute identification of the specific alleles is not 

necessary, adding to the simplicity of analysis. It should be 

noted that this is not a high throughput method. A large 

number of samples would be more appropriately analyzed 

by capillary electrophoresis because of the availability 

of multiplexed reactions and increased sample capacity. 

However, this protocol is appropriate for application to 

small sets of forensic samples as an independent assay to 

support toxicological and other biochemical tests. 

References

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KM, Smerick JB, Budowle B. Validation of Short 

Tandem Repeats (STRs) for forensic usage: Performance testing of fluorescent multiplex STR systems 

and analysis of authentic and simulated forensic 

samples. J Forensic Sci, 2001; 46(3): 647-60.

2. Budowle B, Moretti TR, Niezgoda SJ, Brown BL. 

CODIS and PCR-based short tandem repeat loci: 

Law enforcement tools. From Second European 

Symposium on Human Identification, 1998; 73-

88. Madison, WI: Promega Corp.

3. Masibay A, Mozer TJ, Sprecher C. Promega Corporation reveals primer sequences in its testing kits. J 

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4. Ruitberg M, Reeder DJ, Butler JM. STRBase: A short 

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5. Sullivan KM, Mannucci A, Kimpton CP, Gill P. A rapid 

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14

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