Characterization and validation studies of powerPlex 2.1, a nine-locus short tandem repeat (STR) multiplex system and penta D monoplex.
ABSTRACT In order to increase the power of discrimination for human identification purposes, a nine-locus short tandem repeat (STR) multiplex, the GenePrint PowerPlex 2.1 system (PowerPlex 2.1) developed by Promega Corporation and a separate pentanucleotide-repeat locus, Penta D, were tested. This megaplex system includes the highly polymorphic loci FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01, and D3S1358 and may be used in combination with the eight-locus STR multiplex, the GenePrint PowerPlex 1.1 system (PowerPlex 1.1) that has been previously developed. Three of the loci, TPOX, TH01 and vWA, have been included in both systems for quality control purposes. As with PowerPlex 1.1, PowerPlex 2.1 is also based on a two-color detection of fluorescent-labeled DNA products amplified by polymerase chain reaction (PCR) and provides a valuable tool for accurate and rapid allele determination. The primer sequences used in the PowerPlex 2.1/Penta D system are also presented in this report. To meet the "Quality Assurance Standards for Forensic DNA Testing Laboratories" (FBI), we tested the efficiency and reproducibility of the PowerPlex 2.1/PentaD system by several validation studies that were conducted as a joint project among seven laboratories. Validation tests included concordance studies, sensitivity, and species specificity determination, as well as performance in forensic and environmentally impacted samples. The results produced from these tests demonstrated the consistency and reliability of the PowerPlex 2.1/Penta D system.
- SourceAvailable from: Cynthia Sprecher[show abstract] [hide abstract]
ABSTRACT: Amplification of short tandem repeat (STR) loci has become a useful tool for human identification applications. To improve throughput and efficiency for such uses, the polymorphic STR loci CSF1PO, TPOX, TH01, vWA, D16S539, D7S820, D13S317, D5S818, F13A01, FESFPS, F13B, and LPL have been evaluated, developed, and configured into fluorescently labeled multiplex systems. Eight of these STR loci were combined to generate the PowerPlex System, a two-color multiplex system that supports rapid, accurate, reliable analysis and designation of alleles. The remaining four loci comprise the FFFL System, a one-color multiplex system. The PowerPlex System may be evaluated alternatively as two one-color, four-locus multiplex systems, CTTv Multiplex and GammaSTR Multiplex. The products of multiplex amplification may be analyzed with a variety of fluorescence detection instruments. Determination of genotypes of over 200 individuals from each of three different population/ethnic groups revealed independence of inheritance of the loci and allowed calculation of matching probability, typical paternity index, and power of exclusion for each multiplex.Journal of Forensic Sciences 12/1998; 43(6):1168-80. · 1.24 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Allele distributions for 13 tetrameric short tandem repeat (STR) loci, CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11, were determined in African American, United States Caucasian, Hispanic, Bahamian, Jamaican, and Trinidadian sample populations. There was little evidence for departures from Hardy-Weinberg expectations (HWE) in any of the populations. Based on the exact test, the loci that departed significantly from HWE are: D21S11 (p = 0.010, Bahamians); CSF1PO (p = 0.014, Trinidadians); TPOX (p = 0.011, Jamaicans and p = 0.035, U.S. Caucasians); and D16S539 (p = 0.043, Bahamians). After employing the Bonferroni correction for the number of loci analyzed (i.e., 13 loci per database), these observations are not likely to be significant. There is little evidence for association of alleles between the loci in these databases. The allelic frequency data are similar to other comparable data within the same major population group.Journal of Forensic Sciences 12/1999; 44(6):1277-86. · 1.24 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Alleles of the STR systems HumFES/FPS, HumVWA and HumD21S11 were sequenced and analyzed. Sequence data revealed 3 different systems concerning the complexity of their sequence structure. HumFES/FPS belongs to the STR polymorphism with a simple repeat structure. Only 2 subtypes were found with a base substitution in the 5'-flanking region and no variation in the repeat region. In the STR system HumVWA the sequence structure of the repeat region is more complex, because 2 tetranucleotide units TCTA and TCTG were present. Additionally allele 14 revealed a completely different sequence structure leading to a different electrophoretic mobility. The repeat region of HumD21S11 is compound in structure. The possibility of variation at 3 positions leads to the occurrence of microheterogeneities in fragments of apparent length. In the upper allele range alleles arise with an additional incomplete TA-repeat.Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 02/1994; 106(6):319-23. · 2.69 Impact Factor
Copyright © 2002 by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959.
J Forensic Sci, July 2002, Vol. 47, No. 4
Paper ID JFS2000336_474
Available online at: www.astm.org
Short tandem repeat (STR) loci are small segments of repetitive
DNA sequences three to seven base pairs in length which display
highly polymorphic regions of the human genome (1,2). The small
size of these loci facilitates DNA amplification by the polymerase
chain reaction (PCR) (3,4). Development of simultaneous amplifi-
cation of several such STR loci, known as multiplex PCR (5,6) al-
lows for rapid human identification based on DNA polymor-
phisms. Detection and analysis of multiplexed PCR products may
be conducted on platforms such as capillary (7,8) and flat bed gel
electrophoresis (9) with concordant results. Since very small
amounts of DNA are required even in a highly degraded form
(10,11), this procedure has found many applications in forensic sci-
ences, paternity testing, and other related fields where human iden-
tification is necessary (12–14).
Lins and colleagues (15) have previously reported the develop-
ment of the highly discriminating eight-locus PowerPlex™ 1.1
STR multiplex system. In this study we describe an additional nine-
locus STR multiplex, PowerPlex™ 2.1, and the pentanucleotide-
repeat locus Penta D. The STR loci of these systems can be cur-
rently analyzed with three amplification reactions (one for each of
the PowerPlex™ 1.1, PowerPlex™ 2.1, and Penta D monoplex, re-
spectively) and quality control is monitored by confirmation of
three locus profiles, TPOX, TH01, and vWA, that both multiplex
The PowerPlex™ 2.1 multiplex that is studied here utilizes the
same fluorescent detection system as described for PowerPlex™
1.1 (15) and includes eight tetranucleotide-repeat loci, FGA,
TPOX, D8S1179, vWA, D18S51, D21S11, TH01, D3S1358, and
one pentanucleotide-repeat locus, Penta E. When the PowerPlex™
1.1 system is used, appearance of allele microvariants is rare with
Eleni N. Levedakou,1Ph.D.; David A. Freeman,1Ph.D.; Michael J. Budzynski,1B.S.;
Buddy E. Early,1B.S.; Ruth C. Damaso,2M.S.; Anne M. Pollard,2M.S.; Amy Jo Townley,2M.S.;
Jennifer L. Gombos,2M.S.; Jennifer L. Lewis,2M.S.; Frank G. Kist,3B.S.; Mary E. Hockensmith,3B.S.;
Michelle L. Terwilliger,3B.A.; Elizabeth Amiott,4B.S.; Kevin C. McElfresh,5Ph.D.;
James W. Schumm,5Ph.D.; Suzanna R. Ulery,5M.S.; Felipe Konotop,6B.S.;
Tara L. Sessa,6B.S.; Jeffrey S. Sailus,7M.S.F.S.; Cecelia A. Crouse,6Ph.D.;
Christine S. Tomsey,3M.S.; Jeffrey D. Ban,2B.S.; and Mark S. Nelson,1M.S.
Characterization and Validation Studies of
PowerPlex™ 2.1, a Nine-Locus Short Tandem
Repeat (STR) Multiplex System and
Penta D Monoplex*
ABSTRACT: In order to increase the power of discrimination for human identification purposes, a nine-locus short tandem repeat (STR) multi-
plex, the GenePrint®PowerPlex™ 2.1 system (PowerPlex™ 2.1) developed by Promega Corporation and a separate pentanucleotide-repeat locus,
Penta D, were tested. This megaplex system includes the highly polymorphic loci FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01,
and D3S1358 and may be used in combination with the eight-locus STR multiplex, the GenePrint®PowerPlex™ 1.1 system (PowerPlex™ 1.1) that
has been previously developed. Three of the loci, TPOX, TH01 and vWA, have been included in both systems for quality control purposes. As with
PowerPlex™ 1.1, PowerPlex™ 2.1 is also based on a two-color detection of fluorescent-labeled DNA products amplified by polymerase chain re-
action (PCR) and provides a valuable tool for accurate and rapid allele determination. The primer sequences used in the PowerPlex™ 2.1/Penta D
system are also presented in this report. To meet the “Quality Assurance Standards for Forensic DNA Testing Laboratories” (FBI), we tested the ef-
ficiency and reproducibility of the PowerPlex™ 2.1/Penta D system by several validation studies that were conducted as a joint project among seven
laboratories. Validation tests included concordance studies, sensitivity, and species specificity determination, as well as performance in forensic and
environmentally impacted samples. The results produced from these tests demonstrated the consistency and reliability of the PowerPlex™ 2.1/Penta
KEYWORDS: forensic science, short tandem repeats, PowerPlex™ 1.1, PowerPlex™ 2.1, multiplex, polymerase chain reaction, allele mi-
crovariants, DNA typing, forensics, primer sequences, FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01, D3S1358, Penta D,
CSF1PO, D16S539, D7S820, D13S317, D5S818
1North Carolina State Bureau of Investigation (NCSBI), Raleigh NC.
2Virginia Division of Forensic Science (VDFS), Richmond, VA.
3Pennsylvania State Police (PSP), Greensburg, PA.
4Promega Corporation (PC), Madison, WI.
5The Bode Technology Group (BT), Springfield, VA.
6Palm Beach County Sheriff’s Office (PBCSO), West Palm Beach, FL.
7Charlotte-Mecklenburg Police Dept., Charlotte, NC 28202; formerly at
Texas Dept. of Public Safety, Austin, TX.
*NCSBI has provided most of the Penta D data for this manuscript.
Received 10 Oct. 2000; and in revised form 24 Feb., 28 June, and 20 Dec.;
accepted 21 Dec. 2001, published 7 June 2002.
JOURNAL OF FORENSIC SCIENCES
the exception of the TH01 9.3 allele. With the PowerPlex™ 2.1
system, however, microvariants are frequently observed especially
at the FGA, D21S11, and D18S51 loci (16). Such loci frequently
display microvariant alleles differing from the typical alleles by
one to three base pairs (17,18). Optimal detection and resolution of
microvariants is critical for interpretation of repeat sequences that
are not part of the nominal motif. The pentanucleotide-repeat loci
Penta E and Penta D are effective forensic genetic markers due to
their high degree of polymorphism, low incidence of microvari-
ants, and extremely low stutter. As a result, these loci are highly
discriminating for both single source samples and complex mix-
tures thus aiding in allele interpretation of DNA profiles.
In order to meet the “Quality Assurance Standards for Forensic
DNA Testing Laboratories” (19), a collaborative effort was initi-
ated among seven laboratories to conduct validation studies the re-
sults of which are reported here. Furthermore, since the design and
validation of individual STR primer sets is critical for successful
use on casework evidence, primer sequence data for the Power-
Plex™ 2.1/Penta D system are also presented.
Materials and Methods
The methods used by the reporting laboratories are basically as
recommended in the PowerPlex™ 2.1 System Technical Manual
(20) and are briefly described below. All the procedures were tested
against the NIST Standard Reference Material (SRM 2391a).
Single source samples included blood, saliva, urine, semen, and
vaginal fluids/swabs. For tissue studies, incision scars, ear wax,
fingernail, head and pubic hair, teeth and perspiration were also in-
cluded. Other sources used were non-probative samples and sam-
ples from previously used proficiency tests. Samples that were ob-
tained in dry form were kept temporarily at room temperature until
analysis. Liquid samples were stored at 4°C until stains could be
made. For long term storage, samples were kept at either ?20°C or
?40°C (as stains) or ?80°C (extracted DNA).
Mixture studies included: a) preparations of a series of
DNA:DNA ratios from already quantified samples by the methods
described below, and b) mixtures of body fluids in known volumes
prior to DNA extraction and quantification.
DNA Extraction and Concentration Determination
DNA was extracted using an organic method (21) followed by
DNA purification and concentration through Microcon YM-100
filters (Amicon, Beverly, MA). Alternatively, the FTA Gene Guard
System was used (Life Technologies, Gaithersburg, MD). For se-
men-containing samples, male fractions were separated by a dif-
ferential method (22) followed by organic extraction and concen-
tration with Microcon YM-100 filters.
DNA concentrations were determined using the Quantiblot kit
(Perkin Elmer-Applied Biosystems, Foster City, CA), spectropho-
tometric assay, or agarose yield gels.
One ng of DNA template was used for amplification in a 25 ?L
reaction volume (or alternatively, 0.5 ng in 12.5 ?L) unless other-
wise mentioned, using the reagents provided in the GenePrint®
PowerPlex™ 2.1 System kit including the Gold ST#R 10X reac-
tion buffer (Promega, Madison, WI) and AmpliTaq Gold™ DNA
polymerase (Perkin Elmer-Applied Biosystems, Foster City, CA).
Amplification conditions were performed as recommended in
the PowerPlex™ 2.1 System Technical Manual (Promega, Madi-
son, WI) for the 9600, 9700, or 480 Thermal Cyclers (Perkin
Elmer-Applied Biosystems, Foster City, CA). More specifically
the amplification conditions for the 480 cycler were as follows:
95°C for 11 min, then: 96°C for 2 min, then: 94°C for 1 min, 60°C
for 1 min, 70°C for 1.5 min, for 10 cycles, then: 90°C for 1 min,
60°C for 1 min, 70°C for 1.5 min, for 20 cycles, then: 60°C for 30
min followed by 4°C soak. The program for the 9600 and 9700 cy-
clers was as described above with the exception that the duration of
the 96°C denaturation step as well as each of the cycling steps (de-
naturation-annealing-extension) was reduced in half. With FTA-
extracted samples, the last three amplification cycles were elimi-
nated. Ramp times were as listed in the PowerPlex™ 2.1 System
Technical Manual (Promega, Madison, WI) for all labs except for
NCSBI, where the protocol for the 480 Thermal Cycler was fol-
lowed (ramp times were not used).
Gel Electrophoresis and Detection of Amplified Products
Prior to electrophoresis, the amplification samples were com-
bined with loading buffer and the Internal Lane Standard 600 (ILS)
provided in the PowerPlex™ 2.1 System kit (Promega, Madison,
WI), or with loading buffer alone depending on whether a three- or
a two-color detection, respectively, was desired. The ILS 600 con-
tains 22 fragments of 60–600 bases labeled with carboxy-X-rho-
damine (CXR) for detection at 650 nm. Allelic ladder samples were
also provided in the kit, containing 5?-labeled fragments with either
carboxy-tetramethylrhodamine (TMR) for detection of FGA,
TPOX, D8S1179, and vWA loci at 585 nm, or with fluorescein
(FL) for detection of Penta E, D18S51, D21S11, TH01 and
D3S1358 loci at 505 nm. When the Penta D monoplex was used, it
was accompanied by its own allelic ladder for FL detection. Occa-
sionally Penta D was detected at 585 nm when labeling was with 6-
carboxy-4?, 5?-dichloro-2?, 7?-dimethoxyfluorescein (JOE).
After addition of the loading buffer, the samples were denatured
for 2 min at 95°C and the amplification products were separated
through a 5% Long Ranger™ denaturing polyacrylamide gel
(FMC BioProducts, Rockland, ME), or a 6% Page-Plus for PBSO
(Amresco, Solon, OH), containing 6M urea and 1X TBE. Allelic
ladders were present on every gel for allelic calls. Electrophoresis
was performed at 60 watts for 1 h and 30 min or at 45–50 W for 1
h and 45 min, using the 43 cm long gels (BRL, Bethesda, MD). The
optimized electrophoresis conditions for PBSO were at 60 watts for
1 h and 55 min using the 44.3 cm long gels (BRL, Bethesda, MD).
The gels were pre-run for 10–20 min at the above conditions in or-
der to achieve a surface temperature of approximately 50°C. Fol-
lowing electrophoresis, fluorescent images were detected with the
FMBIO®II Fluorescent Scanner and analyzed by the FMBIO®
Analysis software (1-D gel analysis) and StarCall™ software (Mi-
raiBio Inc., Alameda, CA). As part of the concordance study (at
Charlotte/Mecklenburg Police Department, NC) and for allele con-
firmation (at PSP and TXDPS), samples were also amplified using
the ABI AmpF?STR™ Profiler Plus™/Cofiler™ kits and were an-
alyzed by the ABI PRISM®310 Genetic Analyzer using the Geno-
typer 2.0 software (Perkin Elmer-Applied Biosystems, Foster City,
Stutter Cutoff Determination
Stutter cutoff values were determined by the VDFS laboratory as
follows: 88 samples were analyzed on a total of 5 gels and were ex-
amined for the presence of stutter bands. The total number of alle-
les was as follows: FGA: 166; TPOX: 158; D8S1179: 148; vWA:
149; Penta E: 161; D18S51: 165; D21S11: 174; TH01: 146 and
D3S1358: 150. For heterozygous samples, the analyst looked for
alleles that were at least 8 bp apart so that the stutter of the higher
molecular weight allele would not inflate the OD value of the lower
molecular weight allele. The following numbers of stutter bands
were found for each locus: FGA: 81; TPOX: 23; D8S1179: 72;
vWA: 93; Penta E: 0; D18S51: 24; D21S11: 50; TH01: 23 and
D3S1358: 30. An average optical density (OD) with background
was calculated for the stutter bands and their preceding alleles ob-
served in each locus. Then the percentage of the stutter OD aver-
age: allele OD average was calculated. The standard deviation (?)
of this percentage was determined and a three standard deviation
value (3?) was obtained. The stutter cutoff values are the percent-
ages of stutter OD average: allele OD average at the 3? value, with
a 99% confidence interval.
The ranges of DNA quantities used for amplification in a stan-
dard volume of 25 ?L reaction were as follows:
• Virginia Division of Forensic Science (VDFS): 0.0625 ng,
0.125 ng, 0.25 ng, 0.5 ng, 0.75 ng, 1 ng and 2 ng for five
donors that donated blood and buccal samples each.
• Promega Corporation/Bode Technology Group (PC/BT): 0.1
ng, 0.2 ng, 0.5 ng, 1 ng, 2 ng, 5 ng, 10 ng and 25 ng for the
K562, CCRF-SB, RAJI, KG-1 and IM9 human cell lines. Two
other cell lines, GM 9947A and GM 9948, and one human ge-
nomic DNA sample were tested in the range of 0.1–2.5 ng.
• Pennsylvania State Police (PSP): 0.03125 ng, 0.0625 ng,
0.125 ng, 0.25 ng, 0.5 ng and 1 ng for control K562 DNA and
for three individuals that donated blood/urine, blood/semen,
and blood/saliva, respectively.
• North Carolina State Bureau of Investigation (NCSBI): 0.1 ng,
0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.75 ng, 1 ng, 2.5 ng, 5 ng, and
10 ng for the K562 human cell line.
• Palm Beach County Sheriff’s Office (PBSO): 0.08 ng, 0.15
ng, 0.3 ng, 0.6 ng, 1.25 ng, 2.5 ng, 5 ng, and 10 ng for the K562
To determine species specificity, various DNA quantities from
the following animal and microbial species were amplified in a
standard reaction volume of 25 ?L as follows:
• NCSBI: 1 and 10 ng template DNA were used for amplifica-
tion for cat, horse, partridge, broiler chicken, rabbit, dog,
chicken, deer, bushbaby, African green monkey, Fascicularis
monkey, mouse, gorilla, crested cockatoo, mallard duck, wild
turkey, sheep, pig, cow, lemur, Siwatu Prosimian bushbaby,
rhesus monkey, stumptail monkey, rat, Himalayan brown
bear, Bacillus (cereus, megaterium and subtilis), Micrococcus
luteus, Staphylococcus (epidermidis, capitis, aureus and
hominus), Escherichia coli, Enterobacter aerogenes, Kleb-
siella pneumoniae, Proteus vulgaris, Pseudomonas fluoren-
scens, Serratia marcescens, Chlamydia trachomatis, Neisse-
ria gonnoherra, Enterococcus faecalis, Salmonella cholerae-
suis, Candida albicans, B. marcescens, Aspergillus niger,
Streptococcus somguis and beta-hemolytic Strep. (Group G).
• PC/BT: 0.5 and 5 ng template DNA for rat, mouse, rabbit,
chicken, dog, cow, monkey, orangutan, gorilla, chimpanzee,
Enterococcus faecalis, Escherichia coli, Pseudomonas aerug-
inosa, Staphylococcus aureus, Hepatitis B virus, Human pa-
pilloma virus and Candida albicans.
• PBSO: 1 ng template DNA was used for amplification for
bovine, dog, mouse, rabbit, rat, gorilla, chimpanzee,
orangutan, monkey, cow, frog, shark, sea lion, damsel fish, pig
• VDFS: 1 ng template DNA was used for amplification for ar-
madillo, bear, bovine, cat, chimpanzee, crane, deer, dog, gib-
bon, gorilla, hawk, horse, orangutan, pig, rat and viper.
• PSP: 0.5 ng template DNA was used for amplification for rab-
bit, dog, cat, ferret and baboon.
Environmental Impact Study
Environmental impact on samples was tested by three different
• NCSBI: Various substances were used to soil individual
white, clean cloth pieces of 3 ? 4 in. in size which were sub-
sequently stained with blood and allowed to dry before DNA
extraction. DNA was extracted with the organic method de-
scribed above. Liquid substances were left to dry before blood
staining. The substances tested by this method were the fol-
lowing: silver, black and magnetic fingerprint powder, red
clay, brown dirt, bleach, detergent, perspiration, motor oil,
urine and gasoline. Two known donors provided blood sam-
• VDFS: Twenty-four small pieces of cotton were each soiled
with the following substances: vigorol hairdressing, two dif-
ferent types of lubricant lotion, feminine deodorant spray,
vaginex, static guard, hand cream, isopropyl alcohol, house-
hold cleaner, ammonia, feminine wash, baby oil, mold, heat,
moisture, heat and moisture, super glue, soap, motor oil, lumi-
nol, blackpowder, ninhydrin, redwop fingerprint powder and
bleach. Blood or seminal fluid was then applied on the soiled
cotton. DNA was extracted by the organic method described
above. Six known donors provided body fluids.
• PSP: Blood samples from a known donor were deposited on
several substrates and were left to air-dry overnight prior to
DNA extraction. The following substrates were used: 5 pieces
of broken glass 8 ? 1 mm each, 1 cm2of oily rag, 1 cm2of
dirty tire, 2 cm2of green leaf, 1 cm2of leather shoe, 7 pieces
of wood 8 ? 1 mm each, 1 cm2of denim, 1 cm2of tennis shoe
and 5 pieces of rusty metal 8 ? 1 mm each.
For DNA mixture studies, DNA quantities from two sources
were mixed at various ratios. For fluid mixtures, body fluids from
two sources were mixed at different ratios and stains of the mix-
tures were subsequently made to be processed for DNA extrac-
tion. For both types of mixtures a total of 1 ng DNA was used in
a 25 ?L reaction volume for amplification. The ratios were as
• VDFS: DNA ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8,
1:9 and 0:10 were tested for two DNA mixtures derived either
from blood or buccal samples donated by two individuals.
Also seventeen vaginal plus semen fluid mixtures were pre-
pared (vaginal swabs were spotted with dilutions of seminal
fluid) at approximate ratios of 1:1, 10:1 and 20:1 and com-
pared with seventeen individual standards.
LEVEDAKOU ET AL. • POWERPLEX™ 2.1 VALIDATION 3
JOURNAL OF FORENSIC SCIENCES
• PC/BT: DNA mixture ratios of 100:0, 99:1, 97.5:2.5, 95:5,
90:10, 80:20, 50:50, 20:80, 10:90, 5:95, 2.5:97.5, 1:99 and
0:100, were used in the following human cell line mixtures:
K562?CCRF-SB, GM 9947A?RAJI, and IM9?KG-1.
• NCSBI: DNA ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,
1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18 and 1:19,
were tested for a DNA mixture of two donors.
• PBSO: NIST samples #2 and #3 were used as a DNA mixture
with the following DNA ratios: 100:0, 99:1, 97.5:2.5, 95:5,
90:10, 80:20, 50:50, 20:80, 10:90, 5:95, 2.5:97.5, 1:99 and
0:100. Also, one body fluid mixture of blood plus blood was
tested with volume ratios of 100:0, 95:5, 90:10, 80:20, 50:50,
20:80, 10:90, 5:95, and 0:100.
• PSP: four individuals provided one specimen each for the
preparation of a total of two fluid mixtures, vaginal plus semen
and blood plus saliva, using fluid ratios of 1:20, 1:10, 1:5, 1:3,
1:2, 1:1, 2:1, 3:1, 5:1, 10:1 and 20:1.
Non-Probative and Proficiency Testing
PSP examined the Cellmark 9902, 9903, 9904, Collaborative 99-
512 and CAP FID-B 1999 proficiency tests and 30 mixed source
specimens (8 vaginal plus semen, 2 semen plus blood and 20 semen
plus saliva) where previous PowerPlex™1.1 data were available or
analysis had been performed with Perkin Elmer 310 or 377 Genetic
Analyzers. NCSBI tested nine adjudicated cases where RFLP or
CTT results had been previously obtained, and analyzed six profi-
ciency tests that included CAP FID-A and B 1998, CAP-FID-A
1999 and CAP PI-A, B and C 1998, which had been tested before
with PowerPlex™ 1.1. VDFS tested thirteen non-probative cases
previously analyzed with PowerPlex™ 1.1. PBSO tested eight
cases where CTT and/ or PowerPlex™ 1.1 data were previously
available. PC/BT tested two cases provided by PBSO.
One hundred blood stain samples from convicted offenders pro-
vided by the NCSBI were analyzed by NCSBI, VDFS and PSP.
Twenty-five of these samples were also analyzed by PC/BT and
PBSO/Charlotte/Mecklenburg Police Department (NC) laboratories.
All 25 samples tested in Charlotte/Mecklenburg laboratory were an-
alyzed using the ABI amplification kits and instruments as described
above in the Gel electrophoresis and detection of amplified products.
Also, 80 separate concordant samples were processed between the
PSP regional laboratories in Greensburg and Bethlehem, PA.
Families were studied by PBSO/TXDPS and VDFS as follows:
• PBSO/TXDPS: Blood or buccal swabs were obtained from
members of three different families consisting of: thirty-five
individuals (Family A/PBSO), twenty-two individuals (Fam-
ily B/ TXDPS) and eighteen individuals (Family C/PBSO).
All members of these families were tested with both ABI and
Promega STR multiplex systems.
• VDFS: Buccal samples were collected from the members of
four different families, consisting of five, seven, eight and sev-
enteen members, respectively.
Results and Discussion
PowerPlex™ 2.1 and Penta D Monoplex Characterization
Using PCR technology, forensic DNA typing exploits the poly-
morphic nature of STR sequences in the human genome thus al-
lowing for fast, reproducible results even when only small amounts
or poor quality of DNA samples are available (10,11). The Power-
Plex™ 2.1 and Penta D loci have been selected based on their high
degree of polymorphism and efficient amplification with minimal
artifacts. The multiplex loci used in these studies have been exten-
sively investigated and comply with the “Quality Assurance Stan-
dards for Forensic DNA Testing Laboratories,” effective October
1998 (19), as well as with international standards (23). Analysis of
13 core STR loci is required prior to inclusion in the National DNA
Index System (NDIS) for searching the U.S. National Database of
convicted offender profiles. Lins et al. (15) have previously re-
ported the development of PowerPlex™ 1.1 which included eight
of these loci, CSF1PO, TPOX, TH01, vWA, D16S539, D7S820,
D13S317 and D5S818. In this report we describe the validation of
PowerPlex™ 2.1 which includes the remaining five tetranu-
cleotide-repeat loci that compose the 13 loci core, FGA, D8S1179,
D18S51, D21S11, D3S1358, and an additional pentanucleotide-re-
peat locus, Penta E. Furthermore, we have tested another pentanu-
cleotide-repeat locus, Penta D which can be used separately as a
The PowerPlex™ 2.1 system permits amplification of nine loci
in a single reaction that is analyzed through gel electrophoresis as
shown in Fig. 1 for 10 NIST SRM2391a DNA samples. Detection
is performed using the Hitachi FMBIO®II fluorescent scanner and
is based on the differential labeling of the loci primers. The loci
FGA, TPOX, D8S1179 and vWA are displayed in red, and Penta
E, D18S51, D21S11, TH01 and D3S1358 are displayed in green
(Fig. 1A). Identically labeled allelic ladders (shown flanking every
set of two DNA samples in Fig. 1A) are loaded on every gel for siz-
ing the most common alleles for each locus, both visually and by
StarCall™ software analysis of the digitized images. The internal
lane standard (ILS) is displayed in blue (Fig. 1A). The ILS allows
precise allele position determination by migrating with each indi-
vidual sample through various gel electrophoresis conditions (i.e.,
gel “smiling” effect, bubbles, etc). Color-separated images can be
generated from this multicolor image as shown in black and white
in Fig. 1B, where for every sample each locus is clearly distin-
guished. The image produced using the monoplex Penta D for 11
population database samples is illustrated in Fig. 2.
The characterization of each of the STR loci of the PowerPlex™
2.1 and Penta D locus including chromosome location, locus defi-
nition, repeat sequence and sequence of primers used for amplifi-
cation is displayed in Table 1. In addition, allelic ladder character-
istics such as size range and number of repeats for each ladder
component are shown. The primer sequences have been selected
based on the need to manage Taq DNA polymerase associated ar-
tifacts. Common examples of such artifacts include terminal extra
nucleotide addition (mostly adenine) of PCR-generated fragments
(24,25) and loss of one repeat unit or repeat slippage (26,27) thus
generating a band of lower intensity described as “stutter.” The uni-
formity of the terminal nucleotide addition has been managed by
primer design and addition of a 30 min final extension step of 60°C
to the amplification program (20). Interference of stutter can be
regulated by determination of stutter cutoff values as percentages
of true allele values for each locus and incorporation of the ob-
tained percentages in the StarCall™ software. Therefore, any value
that falls below the stutter cutoff percentage for each locus (shown
in Table 1 as determined by the VDFS and Promega laboratories)
is automatically considered stutter. Penta D is presented as a locus
with very low stutter (?1%, Table 1) followed by Penta E (1–2%),
TPOX (1.8%), and TH01 (2.8%). D8S1179 and FGA follow (5.0%
and 5.3%, respectively) with the rest of the loci exhibiting higher
LEVEDAKOU ET AL. • POWERPLEX™ 2.1 VALIDATION 5
FIG. 1—Fluorescent imaging of amplified DNA products using PowerPlex™ 2.1
One ng from each of 10 genomic DNA samples (SRM2391a samples #1–10, from left to right, respectively), a positive amplification control (CCRF-SB,
lane 17) and a negative amplification control (lane 18), were amplified using the PowerPlex™ 2.1 multiplex and the amplified products were analyzed by
electrophoresis through a 5% Long Ranger polyacrylamide gel. Detection was performed using the Hitachi FMBIO®II fluorescent scanner. Every two
DNA samples are shown flanked by allelic ladders the component sizes of which are displayed in Table 1. For each DNA sample, all nine loci were de-
rived from a single amplification reaction and are represented within one lane. A: Color image displaying: FGA, TPOX, D8S1179 and vWA loci segments
labeled with TMR for detection at 585 nm (red); Penta E, D18S51, D21S11, TH01 and D3S1358 loci segments labeled with FL for detection at 505 nm
(green); the internal lane standard labeled with CXR for detection at 650 nm (blue) with sizes of 60, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300,
325, 350, 375, 400, 425, 450, 475, 500, 550, and 600 bases. B: The color image of panel A was separated into two individual black and white images for
TMR- (left panel) and FL-labeled (right) products.