Laura Guzman

University of Central Oklahoma

Edmond, OK 73034

Doris M. Kupfer

Dennis Burian

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

November 2009

Final Report

Testing MiniSTR Primers for Addition 

to a PCR-Based Forensic Specimen 

Identification Protocol

DOT/FAA/AM-09/21

Office of Aerospace Medicine

Washington, DC 20591

OK-10-0077-JAH

Federal Aviation

Administration
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
i

Technical Report Documentation Page 

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

DOT/FAA/AM-09-21 

4. Title and Subtitle 5. Report Date 

Testing MiniSTR Primers for Addition to a PCR-Based Forensic Specimen November 2009 

Identification Protocol 6. Performing Organization Code 

 

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

Guzman L,1 Kupfer DM,2 Burian D2 

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

 

11. Contract or Grant No. 

1

University of Central 

Oklahoma 

Edmond, OK 73034 

2

FAA Civil Aerospace Medical Institute 

P.O. Box 25082 

Oklahoma City, OK 73125 

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 

Work was accomplished under approved task AM-TOXLAB 

16. Abstract 

An assay has been developed for polymerase chain reaction (PCR)-based human identity testing using the Federal 

Bureau of Investigation’s human Combined DNA Identity System (CODIS) primers. Recent forensic literature 

has identified difficulties using these primers due to amplicon size and the degraded nature of DNA from 

forensic samples. Primers termed mini Short-Tandem Repeat (STR) primers targeted to the same loci as the 

CODIS primers but which have smaller amplicons have been developed. Two of the three miniSTR primer pairs 

examined with our established assay were successfully tested with forensic DNA. This allowed the substitution of 

new primers for detection of a locus, D16S539, which was poorly defined in our assay and the addition of a 

primer pair for a locus, FGA, not previously included. The replacement of the D16S539 CODIS primers with 

the miniSTR primers will provide more accurate results for this locus. The addition of the FGA mini STR 

primers to the core set of tested loci will increase the overall power of discrimination of our assay. 

 

17. Key Words 18. Distribution Statement 

 

 

Genotyping, Polymerase Chain Reaction, miniSTR Document is available to the public through the 

Defense Technical Information Center, Ft. Belvoir, 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 10 

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

iii

Contents

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

Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

 DNA samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Blood and tissue extraction of genomic DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

 Determination of optimum concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

 Testing of previously analyzed DNA samples at optimum primer concentration . . . . . . . . . . . . 2

 Analysis of primers with freshly extracted blood and tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1

Testing MiniSTR Primers for Addition to a PCR-Based Forensic

Specimen Identification Protocol

Introduction

DNA-based analysis of biological samples from fatal

accident sites can serve as an additional and independent

test for sample identification when results from routine

toxicological testing conflict with preliminary sample

identification. An assay has been developed (1, 2) using

primersforeight Combined DNA Index System(CODIS)

short tandem repeat (STR) loci (3), and two genderdetermining loci (4, 5) to analyze samples from aviation

accidents in support of toxicological findings. However,

the D16S539 CODIS primers have been problematic

due to an amplicon size with low resolution in our assay

that utilizes a microfluidics platform, the Agilent 2100

Bioanalyzer, for electrophoretic separation. The size of

the amplicon is at thelimit of resolution for the polymer,

which is best suited to the other, shorter amplicons used

in the assay.

Work reported recently (6, 7) describes a series of

primers designed for the CODIS STR loci that result in

smaller ampliconsizes. Thedifficultyobtaining consistent

polymerase chain reaction (PCR) results with degraded

forensic DNA was the rationale for designing these additionalminiSTR CODIS primers. Bothweaksignals and

allelic dropout had been observed. Using these miniSTR 

primers was shown to give more efficient and consistent

PCR results with degraded samples, potentially, an additional benefit due to the postmortem origin of the

majority of samples used in our lab. 

The purpose of the work presented here was to test

the miniSTR primers for the D16S539 locus and to

test miniSTR primers for two additional loci, FGA and

D21S11, not currently used in our protocol. Thesmaller

amplicons for the miniSTR version of these three loci

suggested that they were in the size range that could be

resolved by our system and the use of two additional

loci would increase the power of discrimination of our

assay. The primer sets weretested to determine optimum

concentration in the PCR reactions. They were then

tested at this optimal concentration against previously

analyzed forensic DNA samples and human blood and

tissue DNA controls. Our results showed that D16S539

and FGA miniSTR primers but not the D21S11 primers

performed well in our assay and could be incorporated

into the core set of loci to increase the overall power of

discrimination of our assay. 

Materials and Methods 

DNA samples

Initial PCR testing was performed with previously

evaluated DNA samples from positive control 2221,

negative control 2222, and forensic DNA samples 2215,

2216, 2217, 2218, 2219, and 2220. Additional samples

used were blood positive control 12423A and forensic

tissue liver sample 03010577, from which DNA was

newly isolated for this study. 

Blood and tissue extraction of genomic DNA

Samples were processed as described in previous

reports (1, 2). Tissue and blood were stored at -20° C. 

Approximately 25 mg of sample 03010577 liver tissue

was minced and processed using the manufacturer protocol with the Qiagen QIAamp DNA MiniKit #51304

(Qiagen; Valencia, CA). DNA was extracted from 200

ul whole blood from control 12423A using thesame kit

with modifications for blood and body fluid as specified

by the manufacturer.

Following DNA extraction, samples were analyzed

using DNA 7500 chips on an Agilent 2100 Bioanalyzer

(Agilent Technologies;Palo Alto, CA)todetermine DNA 

quality. The NanoDrop1000 SpectrophotometerModel

ND-1000,(NanoDrop Technologies;Wilmington, DE)

was used to determine DNA concentration. 

PCR primers and amplification. Table1liststheprimers

used in this study. All primers were synthesized commercially (Integrated DNA Technologies; Coralville,IA). 

PCR reaction setup was for 25 ul final volume. Each

reaction contained 50 ng template, 200-800 nM each

primer,2.5mMdNTPs,0.1% TritonX-100,1x Amplitaq

Gold buffer, and 2.25 units Amplitaq gold. 

A Perkin-Elmer GeneAmp 9600 (PerkinElmer;

Wellesley, MA) was used for PCR according to the

following protocol: one cycle at 95°C for 11 min and

96°C for 1 min;10 cycles at 94°C for 30 sec, 60°C for

30 sec (ramp time 1:08), and 70°C for 45 sec (ramp

time 0:50); 24 cycles at 90°C for 30 sec, 60°C for 30

sec (ramp time 1:00), and 70°C for 45 sec (ramp time

0:50); 1cycle at 60°C for 30 min; and a 4°C hold until

removal of samples.

Detection of a 1:3 dilution in distilled water of

amplification products was performed on an Agilent
2

2100 Bioanalyzer with DNA 1000 chips and Agilent

Expert software version B.02.03.SI307 (Agilent Technologies; Palo Alto, CA). 

Results and Discussion

Initially, four primer concentrations, 200, 400, 600,

and 800 nM, were tested for each of the three primer

pairs using positive control 2221 template. The primers

testedincludedmD16S539,mFGA, andmD21S11,with

an ”m” designating a miniSTR primer pair. 

Determination of optimum concentration

mD16S539: Peak detection on the Agilent 2100 Bioanalyzershowedtheexpectedpeaksizesfor allmD16S539

primer concentrations. However,the 200 nMconcentration reaction result was below our lower confidencelimit

of 20 fluorescence units (FU). Almost equivalent results

were seen at 400, 600, and 800 nM (Figure 1A), showing that peak intensity did not increase dramaticallywith

increasedconcentration. Therewas goodpeak resolution,

low primer dimer, and low secondary product produced

at all primer concentrations tested (Figure 1A). 

mFGA: The Agilent 2100 Bioanalyzerelectrophoresis

results displayed positive results for all FGA primer concentrations. However,the200nMconcentrationshowed

peaks below our lower confidence limit of 20 FU. Best

results were seen at 400 and 600 nM (Figure 1B). There

was good peak resolution and low primer dimer product

at these concentrations. Secondary product increased

with concentration, and at 800 nM, a significant primer

dimer and secondary product was seen (See Figure 1B). 

mD21S11: Noprimer concentrationsshowedpositive

results with PCR. Due to the negative result at all four

primer concentrations using our established laboratory

protocol, no furtherworkwas donewith this primer pair. 

For mD16S539 and mFGA, a concentration of 400

nM was judged to be optimal, based on maximal specific

product peak heights combinedwith minimal secondary

product formation. This concentration was used in the

additional testing performed next.

Testing of previously analyzed DNA samples at 

optimum primer concentration 

The three miniSTR primer pairs were tested against

DNA fromsix forensic samples,2215-2220,thathadbeen

characterized in our laboratory. Our previous results had

determined that Sample 2215 and Sample 2219 came

from the same source, Sample 2216 and Sample 2218

camefromthesamesource, and Sample2217and Sample

2220 camefromthesamesource. The CODIS D16S539

primersetwasincludedfor comparisonpurposeswiththe

miniSTR D16S539primers. Wehadpreviously observed

poor peak resolution from the CODIS primer set on the

Bioanalyzer due to sizes of the amplicons. Using both

primer sets for locus D16S539 in this part of the study

allowed a direct comparison.

D16S539:Primersshowedresultssimilartothosefrom

the previous analysis. Results showed single peaks for all

samples. Product sizes are listed in Table 2. Examination

oftheelectrophoresisresults only allowed discrimination

of samples 2215, 2216, 2218, and 2219 from samples

2217 and 2220. These results were consistent with our

previous results for this locus but inconsistent with the

original analysis reported from eight loci without the

inclusion of the D16S539 results. 

mD16S539:PCR productsmigratedatsizessomewhat

larger, 115-127 bp, than thereported range of 81-121 bp

(6). There was no primer dimer present, but secondary

product was seen for all samples. These primers provided

better peak resolution than the CODIS D16S539 primers presumably due to the smaller size of the products. 

Samples 2215, 2216, 2218, and 2219were heterozygous;

Samples 2217 and 2220were homozygous. Peak sizes are

listed in Table 2. Overlays of the electrophoresis results

showed that Samples 2215 and 2219 had the same pair

Table 1. Primers used in study.

Locus Primer Sequence

Product Size 

Range (bp) Reference

Size 

Reduction 

(bp)

D16S539 GGG GGT CTA AGA GCT TGT AAA AAG 264-304 8

GTT TGT GTG TGC ATC TGT AAG CAT GTA TC

D16S539 ATA CAG ACA GAC AGA CAG GTG 81-121 6 152

(miniSTR) GCA TGT ATC TAT CAT CCA TCT CT

D21S11 ATT CCC CAA GTG AAT TGC 153-211 6 33

(miniSTR) GGT AGA TAG ACT GGA TAG ATA GAC GA

FGA AAA TAA AAT GCA TAT TTA CAA GC 125-281 6 71

(miniSTR) GCT GAG TGA TTT GTC TGT AAT TG

Size reduction is comparison of the miniSTR to the corresponding CODIS primer-generated amplicon
3

Figure 1. Overlays of PCR products from miniSTR primer titration series using positive control DNA

2221. 800 nM (Red), 600 nM (blue), 400 nM (green), 200 nM (turquoise). FU Fluorescent Units. A. 

mD16S539 PCR products. B. mFGA PCR products.
4

of alleles; Samples 2216 and 2218 shared thesame alleles,

and Samples 2217 and 2220 shared the same allele (data

not shown). This is consistent with previous results for

other loci used to test these samples. 

Most important was theincreased peak resolution for

Sample 2216 with the mD16S539 primers. Electrophoresis of product from the CODIS primer pair (Figure

2A) shows a poorly resolved doublet that could be easily

interpreted as a single peak from a homozygote. Due to

theshorter ampliconlengthsfrommini-STR primerpair,

the doublet is easily resolved and the true result emerges

(Figure 2B).

FGA: PCR products were within the reported range

of 125 - 281 bp (6) for all samples. Peak resolution and

intensityweregoodfor allsamples. Verylittleprimerdimer

or secondary product was seen in all cases. All samples

were heterozygous and showed product sizes as seen in

Table2. Afterexamining theelectrophoresisresults,itwas

determined that Samples 2215 and 2219 had the same

alleles, Samples 2216 and 2218 had thesame alleles, and

Samples 2217 and 2220 had the same alleles. This was

consistent with previous results, as noted above. 

Table 2 shows a summary of the results for this

analysis. mD16S539 primers gave products that were

better resolved on the Bioanalyzer DNA1000 chip than

were the CODIS D16S539 primers and led to the correct interpretation of the sample relationships. While

CODIS D16S539 primer products suggested broad

homozygous peaks for Samples 2215, 2216, 2218, and

2219, mD16S539 products for the same samples were

clearly heterozygous peaks on the same DNA1000 bioanalyzer chip. Consistent results with the mFGA primer

set suggested that replacement of the CODIS D16S539

primer pair with the mD16S539 primers and addition

Table 2. PCR product sizes for the primer pairs tested with six forensic samples. 

Loci Peaks

Sample 1 

2215

Sample 2 

2216

Sample 3 

2217

Sample 4 

2218

Sample 5 

2219

Sample 6 

2220

Sample 

Comparison

mFGA 1 147 148 1=5, 2=4, 3=6

2 154 154 155 155

3 160 157 158 162

4 168 169

D16S539 1 290 292 1=2=4=5, 3=6

2 295 295 296 295

mD16S539 1 118 118 119 119 1=5, 2=4, 3=6

2 121 122 123 123

3 125 126

basepairs

A. B.

Doublet

295 bp 122, 125 bp

CODIS D16S539 miniSTR D16S539

Figure 2. Comparison of CODIS D16S539 and miniSTR D16S539 for Sample 2216. Basepairs (bp). FU 

Fluorescent Units. A. Results from CODIS D16S539 primers showing a single broad peak with poorly defined 

doublet (arrow), B. miniSTR D16S539 showing well-defined doublet.
5

of the mFGA primer pair would dramatically increase

the power of discrimination. 

Analysis of primers with freshly extracted blood 

and tissue

DNA was extracted from forensic blood and tissue

samples (Materials and Methods) to assurethat template

newly isolated from degraded forensic samples could be

usedastemplatewiththetwominiSTR primersets,mFGA 

and mD16S539. DNA 7500 chip results indicated the

tissue DNA was more degraded than blood DNA (See

Figure 3). The quality of both samples is consistent with

previously observed genomic DNA isolations performed

with the method described in Materials and Methods

(Datanotshown). Both templateswere used inPCR with

mFGA, mD16S539, and CODIS D16S539 primers.

CODIS D16S539: Results for this analysis indicate

good peak intensity (tissue 30 FU, blood 90 FU), with

little primer dimer or secondary product (tissue 7 FU 

and blood 2 FU). Blood DNA showed a homozygous

peak at 288 bp and tissue DNA was heterozygous, with

peaks at 289 bp and 296 bp. 

mD16S539: Primers showed positive results with

one peak appearing beyond the expected range of 81

bp-121 bp for blood DNA. Peak intensities were good

(tissue 50 FU and blood 145 FU) and no primer dimer

was detected. A secondary product was present but was

below our significance cutoff of 20 FU (tissue 14 FU and

blood 3 FU). Blood DNA was homozygous, with a peak

at 118 bp; tissue DNA was heterozygous, with peaks at

118 bp and 126 bp. 

mFGA: Primers showed positive results within the

expected range of 125- 281 bp for all samples. Peak

intensities were acceptable (60 FU for tissue, 80 FU for

blood) with little primer dimer present for all samples. 

However, secondary product was high in blood, slightly

above our significance cutoff of 20 FU (8 FU tissue, 24

FU blood). Blood DNA showed heterozygous peaks of

150bpand157bp, andtissue DNA showedheterozygous

peaks of 151 bp and 161 bp. 

Although results for the CODIS D16S539 primers

were the same as the miniSTR primers, mD16S539

results still showed increased peak resolution. Regarding

the templates used for this portion of the study, the difference in degradation of the DNA (Figure 3) appeared

to have little effect on the PCR outcome except that the

signal in tissue was lower than in blood. 

There were three concerns with the findings in this

study. First,thefailureofthemD21S211primerssuggests

that none of the primer concentrations used in this study

wereideal underthe cycling conditions used. Second,the

secondaryproductpeaksseeninthemFGA PCR products,

Figure 3. Electropherogram comparing DNA isolates from forensic whole blood (red) and liver (blue) with the 

liver sample showing overall smaller species. FU Fluorescent Units. Negative control (green). 

Overlay of DNA isolated from blood or tissue

Low MW Marker High MW Marker
6

while not unique to this locus, preclude multiplexing of

reactions in a single tube. This is not a significant issue

for the single locus analysis we currently use since the

expected product size range is known, therefore causing

little difficulty determining the primary product. However, in a multiplex reaction, secondary products may

fall within the expected size ranges of other primer pairs

resulting in multiple peaks, thus making correct data

evaluation difficult. Theresultsseen for bothmD21S211

and mFGA suggest that additional primer concentration

optimization would be useful. Finally, the higher-thanexpected product sizes (seven basepairs larger than the

published size) we see with the mD16S539 primers in

samples 2216 and 2218 are most likely due either to an

error in the sizing algorithm or a migration shift resulting from the matrix used with the bioanalyzer. We have

seen this to different extents with other CODIS primers; however, because our assay is based on comparative

analyses between samples and does not rely on absolute

alleleidentification,thisdoesnotinterferewithour ability

to arrive at valid conclusions.

Conclusions

miniSTR D16S539hassmaller amplicons andprovides

dramatically better peak resolution over the CODIS 

D16S539 PCR products. The improved resolution increases the power of discrimination at this locus because

n+1 heterozygotes can now be detected. The example

provided here demonstrated that the level of resolution

with the CODIS D16S539 amplicon is of such poor

quality as to lead to incorrect comparison conclusions. 

Therefore, the miniSTR version will be substituted into

our assay in the future, and miniSTR FGA will also be

incorporated into the core set of loci used at CAMI, further increasing the power of discrimination of our assay. 

Reducing the size of amplicons by using the miniSTR 

primers proved to be useful in enhancing discrimination,

retrieving a locus that had performed poorly in our assay

and allowing us to incorporate an additional locus for

whichthe CODIS versionhadaPCR productbeyondthe

lowerlimitofdetectionforn+1heterozygotesinour assay. 

We have recently converted to the use of a robot for

setup of our genotyping reactions. With the use of this

instrument, the need for uniform primer concentrations in our reactions is decreased, since the increase in

complexity of reaction setup can be easily programmed. 

This suggests to us that in the future, we could explore

additional miniSTR primers, including reinvestigating

the use of mD21S11. 

Additionally, it should be noted that thesamples used

in this assay areforensic and degraded to various degrees. 

Smaller amplicons may be particularly beneficial when

handlingthedegraded DNA foundinforensic samples. To

date, we have not noticed samples which are completely

PCR-resistant using our core CODIS set of primers. 

However,theexistenceof additionalminiSTR primersets

may be useful in the future if we analyze larger numbers

of heavily degraded forensic samples. 

References

1. Kupfer D.M., Huggins M., Cassidy B., et al. (2006). 

A rapid and inexpensive PCR-based STR genotyping method for identifying forensic specimens. 

Washington, DC: Office of Aerospace Medicine

Report no. DOT/FAA/AM-06-14, 1-14.

2. Kupfer D.M., Jenkins M., Burian D., Canfield D.V. 

(2008). Use of alternative primers for gender

discrimination in human forensic genotyping. 

Washington, DC: Office of Aerospace Medicine

Report no. DOT/FAA/AM-08-8, 1-5.

3. Budowle B., Moretti T.R., Niezgoda S.J., Brown B.L. 

(1998). CODIS and PCR-based short tandem

repeat loci: Law enforcement tools. Publication

number 98-06 of the Laboratory Division of the

Federal Bureau of Investigation.

4. Aasen E., Medrano J.F. (1990). Amplification of

the ZFY and ZFX genes for sex identification in

humans, cattle, sheep and goats. Biotechniques 8,

1279-81.

5. McKeown B., Stickley J., Riordan A. (2000). Gender

assignment by PCR of the SRY gene: An improvement on amelogenin. Progress in Forensic Genetics 

8, 433-5.

6. Butler J.M., Shen Y., McCord B.M. (2003).The development of reduced size STR amplicons as tools

for analysis of degraded DNA. Journal of Forensic 

Sciences, 48, 1054- 64.

7. OpelK.L., Chung D.T., Drabek J.,ButlerJ.M. (2007). 

Developmental validation of reduced-size STR 

miniplex primer sets. Journal of Forensic Sciences,

52,1263-71.

8. Krenke B.E., Tereba A., Anderson S.J., et al. (2002). 

Validation of a 16-locus fluorescent multiplex

system. Journal of Forensic Sciences, 47, 773-85.