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.
JOURNAL OF FORENSIC SCIENCES
values such as vWA (7.9%), D18S51 (8.3%), D21S11 (9.2%) and
D3S1358 (9.7%). Similar data were obtained previously as re-
ported in Promega Technical Manual (20). Appearance of stutter
bands becomes an important issue when mixtures of samples are
being analyzed that contain bands of variable intensities. In such
cases, interpretation may rather be based on analyst’s discretion
and experience by visual examination of the profiles than on stut-
ter values incorporated in the StarCall™ software. Along these
terms, the importance of highly polymorphic loci with low stutter,
such as Penta D and E, becomes apparent in resolving controver-
Another feature characteristic for some loci of PowerPlex™ 2.1
such as FGA, D21S11 and D18S51, is the frequent presence of mi-
crovariant alleles or alleles with sizes greater than the typical allele
size by one, two or three bases (17,18). To achieve accurate allele
determination in such cases, the allelic ladders for these loci have
been supplemented with bands of one half repeat size. The ILS was
also utilized so that allele calls may be based on both sizing systems.
Appropriate adjustments may also be necessary when using the
StarCall™ software to allow for precise size evaluation of such mi-
crovariants. The user may vary the allele call window for each lo-
cus as he desires, so that when in a particular locus microvariants are
frequently flagged “out of range” and the used window is, for ex-
ample, 1 bp (?1.00 to ?1.00), a tighter window may be chosen for
that locus to allow for the appropriate allele call (such as a ?0.50 to
?0.49 bp range) which will reduce the “out of range” calls.
LEVEDAKOU ET AL. • POWERPLEX™ 2.1 VALIDATION 7
FIG. 2—DNA profiling using the Penta D monoplex
Genomic DNA from eleven population database samples (325–335) was amplified using the Penta D monoplex and the amplified products were ana-
lyzed by electrophoresis through a 5% Long Ranger polyacrylamide gel. Detection was performed using the Hitachi FMBIO®II fluorescent scanner at 505
nm for FL-labeled products. Repeat sizes of the Penta D allelic ladder are shown at the first lane from left (2.2, 3.2, 5, 7–17); (?): negative amplification
control (amplification reagents without DNA), (?): positive amplification control (DNA from the K562 cell line) and N: negative DNA extraction control
(extraction reagents without blood-stained sample).
JOURNAL OF FORENSIC SCIENCES
FIG. 3—Sensitivity of PowerPlex™ 2.1/Penta D
Various amounts of DNA templates were used for amplification, the amplified products were separated in a denaturing 5% Long Ranger polyacrylamide gel and analyzed
by the Hitachi FMBIO®II fluorescent scanner. A: The DNA profiles are derived from K562 and CCRF-SB cell lines using for amplification 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0
and 25.0 ng of genomic DNA from each cell line. B: K562 DNA was tested for Penta D in quantities of 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 2.5, 5.0 and 10.0 ng. (?): negative
LEVEDAKOU ET AL. • POWERPLEX™ 2.1 VALIDATION 9
Sensitivity of the system was tested by amplification of various
DNA quantities ranging from 0.03125 ng to 25 ng (per 25 ?L reac-
tion). All samples tested at PC showed complete profiles at DNA
template quantities as low as 1 ng. Also, amplification of most loci
was noted in almost all samples at PC even at quantities of 0.2 and
0.1 ng. For the reference sample K562, as shown in Fig. 3A(data pro-
vided by PC), at 0.5 ng of K562 DNA, one of the FGA alleles (upper
band, allele 24) has dropped out. At 0.1 ng K562 DNA other loci have
started to drop out (Penta E, D18S51, and D21S11). In contrast, the
CCRF-SB cell line gave detectable results in all loci even at the low-
est DNA quantity tested (0.1 ng). NCSBI and PBSO results were in
agreement with the results obtained by PC for the K562 DNA (Table
2). PSP reported allelic dropouts mostly at Penta E, FGA and
D18S51, at DNA quantities as low as 0.0625 ng, 0.125 ng and 0.5 ng
depending on the sample examined. Tests performed at VDFS
showed allelic dropout at all quantities below 0.5 ng. Amplification
for Penta D locus was observed even at 0.1 ng of DNA template (Fig.
3B, NCSBI data). It is noteworthy that in those situations that some
loci failed to amplify, there was still a substantial amount of infor-
mation that could be obtained from the remaining loci. It also appears
that sensitivity may be template dependent, as for example, in the
K562 cell line where the apparent difference of intensity between the
two FGA alleles resulted in a switch of the sensitivity threshold from
0.1 to 0.5 ng DNA. The allelic FGA imbalance in the reference sam-
ple K562 provides for a useful positive control of amplification of
the weaker bands since it is possible that samples in question to ex-
hibit a similar locus imbalance. A summary of the sensitivity results
is illustrated in Table 2 which indicates that allelic dropouts may be
observed below 0.5–0.6 ng of DNA template. Taking into account
all the results derived from the participant laboratories an optimal
range of 0.75 to 2 ng DNA was determined to be used for amplifica-
tion. Nevertheless, this does not imply that complete profiles cannot
be successfully produced at very low DNA amounts as it was occa-
sionally noticed. In such situations, one must have confidence in the
obtained results especially when full profiles are generated. This ob-
servation is particularly useful in forensic casework where fre-
quently only limited evidence material is available and there is no op-
tion of using the optimal DNA amount. We have also noticed that
when DNA quantities of 5 ng or greater are amplified, background
level as well as stutter may increase and an imbalanced profile among
loci may be observed as a result of better amplification of smaller loci
as compared to the larger ones.
Consistency Among Tissues
To determine that the allelic profile remains the same from tis-
sue to tissue obtained from a single individual, two studies were
performed where samples from various tissues such as blood,
saliva, vaginal fluid, semen, head hair, fingernail, ear wax, urine
and incision scar (VDFS, five donors), or blood, saliva, vaginal
fluid/semen, perspiration, teeth and head and pubic hair (PSP, four
donors) were tested. All tissues obtained from the same donor gave
identical profiles (data not shown). These results demonstrate that
regardless of the tissue tested, each individual is characterized by a
single allele profile for all the loci of PowerPlex™ 2.1.
In order to determine if bacterial, fungal, or animal DNA could
yield amplified products from human-derived STR primers, ex-
tracted DNA from a panel of microbial and animal species was
tested. Penta D locus showed no amplification products in any mi-
crobial and animal species tested at the NCSBI. For all the micro-
bial species studied at the NCSBI and PC/BT, amplification of
PowerPlex™ 2.1 STR alleles was negative as well. Negative am-
plification results were also obtained for all the non-primate sam-
ples tested by all the reporting laboratories. For the higher primates
a DNA banding pattern was observed with the majority of alleles
migrating off ladder or between ladder bands. The data for the pri-
mates are summarized in Table 3 which indicates the loci where
amplification is likely to be observed for each species. It should be
noted, that use of monoplexes may identify the locus that each al-
lele is assigned to. More specifically: For monkey, amplification
was noted at Penta E, FGA and TPOX (NCSBI: Stumptail and Fas-
cicularis monkey), or only at Penta E and FGA (PC/BT). For
orangutan, bands were observed at Penta E, TH01 and FGA
(VDFS); at Penta E, TH01, D3S1358 and FGA (PC/BT); and at ev-
ery locus except TPOX, D8S1179 and vWA (PBSO). For gorilla,
bands were seen at all loci (PBSO, PC/BT), except at TH01, TPOX
JOURNAL OF FORENSIC SCIENCES
and D8S1179 (NCSBI), or D21S11 and D8S1179 (VDFS). For
chimpanzee amplification was also seen at all loci (PBSO), except
at Penta E (PC/BT), or D3S1358 and D8S1179 (VDFS). For ba-
boon, amplification was noticed at Penta E, FGA and TPOX (PSP),
and for gibbon at TH01, FGA and TPOX (VDFS).
In conclusion, amplification products were not obtained in mi-
crobial and non-primate animal species. From the primates tested,
the majority exhibited alleles migrating between ladder bands and
off ladder and in several occasions more than two bands per locus
were noticed. It should also be mentioned that no individual pri-
mate showed allele bands with the appropriate number of alleles or
sequence lengths for all 10 loci, in a manner characteristic for hu-
Environmental Impact Study
These studies were designed to determine the efficiency and ac-
curacy of the PowerPlex™ 2.1/Penta D system when biological
samples have been exposed to different types of environmental in-
sults. Therefore, a variety of such substances, both liquid and solid,
were tested by contaminating samples of donors with known pro-
files using different experimental approaches. It was observed that
regardless of the DNA source, the contaminant, or the method used
(substrate directly stained with blood, or substance-soiled cloth and
subsequently blood-stained), the loci that successfully amplified
produced profiles that matched their respective standards. PSP re-
ported amplification in all loci for all the substrates tested. For the
contaminants tested by NCSBI and VDFS, amplification was no-
ticed for all loci with the exceptions of allele dropouts for the con-
taminants shown in Table 4. For mold, or heat plus moisture am-
plification failed in all loci (VDFS). Also samples contaminated
with hand cream amplified only in vWA and TH01 loci (VDFS).
Penta E dropouts were also noticed when moisture, super glue, mo-
tor oil and bleach (data from VDFS), or red clay and perspiration
were tested (NCSBI data). From the VDFS studies it was noticed
LEVEDAKOU ET AL. • POWERPLEX™ 2.1 VALIDATION 11
JOURNAL OF FORENSIC SCIENCES
that TPOX was also lost when super glue and motor oil were tested.
Amplification in the Penta D locus remained unaffected by all the
substances tested by the NCSBI laboratory (data not shown). From
these studies we concluded that DNA typing may be successfully
produced using the PowerPlex™ 2.1 and Penta D loci even under
circumstances that might be considered at first as inhibitory, such
as the in presence of detergents, bleach, gasoline, alcohol, soap,
ammonia or household cleaners. More importantly, other than the
observation that a few loci failed to amplify in the presence of some
of the substances, the obtained DNA profile remained unaltered.
These results confirm previous studies on the effect of various en-
vironmental insults on DNA typing by either RFLP (28), or PCR
Mixture studies were conducted to determine the efficiency of
DNA amplification from two sources at different ratios. The im-
portance of testing the sensitivity of the PowerPlex™ 2.1/Penta D
system in mixtures lies in the fact that often a single forensic sam-
ple may contain DNA from two or more individuals at unpre-
dictable ratios and the profiles need to be correctly identified. In
these situations, information from as many loci as possible is de-
sirable. DNA mixtures and body fluid mixtures were used at vari-
ous ratios of the mixture components. For each mixture, a cutoff ra-
tio was determined below which partial or no profile was detected
from the source with lower DNA concentration.
DNA mixtures: The PC/BT laboratory reported a cutoff ratio of
20:80 for the mixtures of K562?CCRF-SB (Fig. 4A), GM
9947A?RAJI and IM9?KG-1 (data not shown). Nevertheless, as
shown in Fig. 4A for the mixture of K562?CCRF-SB some loci of
the sample with lower DNA concentration showed amplification in
lower ratios tested, such as FGA, D8S1179, vWA and D21S11 for
the K562 sample. The cutoff ratios reported for the DNA mixtures
tested by VDFS were 20:80 and 30:70. NCSBI reported a cutoff ra-
tio of 1:10 whereas several loci of the low DNA concentration sam-
ple showed amplification even at the lowest ratio tested (1:19), as
for example Penta D (Fig. 4B). PBSO tested a DNA mixture that
showed a cutoff ratio of 20:80.
Body fluid mixtures: VDFS typed correctly the profiles of the
vaginal plus semen mixtures tested as compared with their respec-
tive standards, when the ratio of 1:1 was used. When greater dilu-
tions of seminal fluid used at VDFS (10 or 20 times more dilute),
partial or complete dropout of the sperm fraction profile was ob-
served. PSP tested two mixtures of vaginal plus semen and blood
plus saliva and whereas in the vaginal plus semen mixture all loci
of the sperm fraction were detected at all ratios examined (1:20 to
20:1), in the blood plus saliva mixture the cutoff ratio was 1:2.
Among all ratios tested at PBSO (100:0 to 0:100) for a blood plus
blood mixture, complete profiles of both sources were reported at
a ratio of 50:50.
In summary (Table 5), DNA mixtures with ratios between 1:10
and 30:70 gave complete profiles of both donors at all loci tested,
whereas body fluid mixtures exhibited efficient ratios between 1:2
and 1:1. The difference in the ratios observed among the mixtures
could partly be attributed to the fact that each mixture is unique in
terms of biochemical composition and size of its allele compo-
nents. Consequently, amplification rates may vary depending on
the combination of the alleles that are present in each mixture. In
addition, body fluid ratios generated by volume to volume mixing
of different body fluids, may approximate the actual ratios of their
DNA contributors. In forensic mixture samples, one doesn’t know
either the relative concentrations of the DNA donors, or the volume
ratios of body fluids. For that reason, the mixture experiments
served as a guide to determine the theoretical amplification expec-
tations of the multiplex, since they only mimic actual forensic mix-
tures. As it was demonstrated from these studies, the multiplex suc-
cessfully identified potential donors in mixtures similar to the ones
seen in forensic samples.
To confirm the reliability of the PowerPlex™ 2.1 System within
each individual laboratory, specific samples were re-analyzed. The
results of the PowerPlex™ 2.1 system testing were compared to
previous results obtained from testing by other analysts or other ap-
proaches, such as RFLP, CTT, PowerPlex™ 1.1, etc, or by using
different instrumentation (Hitachi FMBIO system versus ABI tech-
nology). The samples examined were from non-probative cases,
old proficiency tests, and body fluid mixtures as described in Ma-
terials and Methods section. In every laboratory, the allele typing
based on PowerPlex™ 2.1 system was consistent regardless of the
analyst or instrumentation and confirmed interpretations made on
previously analyzed non-probative cases.
The consistency of the PowerPlex™ 2.1 multiplex was validated
among three laboratories (NCSBI, VDFS, and PSP) through a con-
cordance study of 100 convicted offender samples obtained from
the NCSBI DNA database (data not shown). The PC/BT and
PBSO/Charlotte/Mecklenburg laboratories analyzed the first 25 of
these samples. All the samples were typed correctly and in agree-
ment among the laboratories with the following exceptions: a 22.3
FGA allele which was typed as 22.2 or 23, a 21.2 D18S51 allele
which was typed as 22 and one FGA allele that four laboratories re-
ported it as a 22.2 and one laboratory reported it as a 22.3. After re-
analysis of these samples, all laboratories reported the same results
with the exception of the last FGA allele that remained a non-re-
solved discrepancy (22.2 or 22.3). It should be noted that all three
samples involved microvariant calls and the discrepancies were no
more than one to two base pairs apart. Such allele calls that ap-
proach the resolution and detection limits of the system may be
subjective and reflect slight experimental variations among labora-
tories. Such factors include length of electrophoresis, gel migration
artifacts, sample quantity loaded on the gel, use or not of ILS, and
sizing window settings in the StarCall™ software to allow for the
microvariant definition. Minor discrepancies of this kind should
not affect CODIS searches since CODIS matching algorithms al-
low for a high stringency match when there is a 1 bp difference be-
tween two alleles. Also, CODIS searching parameters allow for
moderate stringency searches that can return for example, a 12 lo-
cus match when two 13 loci samples differ in one locus. Such a
match will draw the analyst’s attention for further examination and
analysis of these samples.
To test the compatibility of the PowerPlex™ 2.1 system with the
ABI technology, STR profiles of the 25 samples analyzed at the
Charlotte/Mecklenburg laboratory, were obtained using the ABI
amplification and analysis systems. The results were in concor-
dance with the PowerPlex™ 2.1 data returned from the other labo-
PBSO and TXDPS analyzed three families consisting of several
generations to determine if there were parent/child STR allele
LEVEDAKOU ET AL. • POWERPLEX™ 2.1 VALIDATION 13
FIG. 4—Performance of PowerPlex®2.1/Penta D in DNA mixture studies
DNA samples mixed in various ratios were used for amplification, the amplified products were separated in a denaturing 5% Long Ranger polyacrylamide
gel and analyzed by the Hitachi FMBIO®II fluorescent scanner. A total of 1 ng of DNA was used for amplification. A. DNA mixture of K562 and CCRF-SB
cell lines with 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. B. DNA samples from two donors (A and
B) were mixed at ratios: 1:1 (1), 1:2 (2), 1:3 (3), etc. to 1:19 (19), and the mixtures were analyzed for Penta D. (?): negative amplification control. (K562):
positive amplification control.
JOURNAL OF FORENSIC SCIENCES
transfer mutations. Three generations of Family A consisting of 35
individuals, four generations of Family B consisting of 22 individ-
uals and three generations of Family C consisting of 18 individu-
als, were analyzed for the 13 core STR loci. Three parent/child mu-
tations were detected in Family A and three parent/child mutations
in Family B, but no mutations were detected in Family C. As shown
in Table 6, mutations were observed for three individuals at
D21S11, one individual at CSF1PO, one individual at D8S1179
and one individual at D7S820 (offspring mutations). Also the
parental allele which is most likely the contributor for each of these
mutations is also indicated in Table 6. All alleles were verified us-
ing both ABI and Promega STR multiplex systems. Data obtained
from the VDFS laboratory include four families consisting of at
least five members each. In one of these families one mutation was
observed where an FGA 22 or 25 allele from the father was possi-
bly mutated to a 21 in his son’s profile (Table 6, Family D). Se-
quencing of the amplified products in these studies may determine
the origin of these mutations. Generally, single-step mutations ac-
count for approximately 90% of STR mutation events (30). These
data have demonstrated that mutations can occur from a parent to a
child and may provide information to paternity laboratories where
current practice suggests that a difference in two or more loci of the
DNA profiles should be observed before an alleged parent could be
eliminated as a biological parent. Mutation rates vary at each STR
locus as it has been currently estimated (30–32) and they are ap-
proximately on the order of 1.2 ? 10-3per locus per gamete per
generation, with the FGA locus exhibiting a four-fold higher rate of
mutation (4.01 ? 10-3). To confirm mutation rates, a greater num-
ber of families need to be examined. Generally, mutation rates do
not affect casework interpretation of analysis of forensic evidence
samples using STRs because comparisons are being made between
samples either originating (in the case of an inclusion) or not orig-
inating (in the case of an exclusion) from the same source.
The validation studies presented in this report demonstrate that
the fluorescent PowerPlex™ 2.1 multiplex STR system is a sensi-
tive, reliable forensic PCR DNA typing technique. PowerPlex™
2.1 has been shown to be species-specific and robust when samples
have been exposed to environmental insults and contaminants. Re-
gardless of sample preparation protocols, interlaboratory and in-
tralaboratory genotypic comparisons show great concordance. De-
tection and allele designation of microvariants is confidently
determined using the internal lane standard and appropriate win-
dow sizing in the StarCallMTsoftware.
Through population database studies performed by NCSBI,
PBSO, VDFS, and PC/BT, the multiplex was demonstrated to be
highly discriminating (33). The PowerPlex™ 2.1/Penta D system
and the STR loci primer sequences presented here facilitate suc-
cessful application in the forensic field.
The authors would like to thank the forensic scientists Jeff
Fumea and Barbara Flowers from the PSP laboratory as well as De-
bra Glidewell and Sandra Gibson from the PBSO laboratory for
their contributing efforts to these studies. Also, Cynthia J. Sprecher
from the Promega laboratory for her valuable assistance in the re-
viewing process of the manuscript.
Felipe Konotop is a Visting Scientist in the Palm Beach County
Sheriff’s Office Serology/DNA Section. Mr. Konotop was pro-
vided a scholarship from the laboratory of Dr. Elizeu Fagundes de
Carvalho in the Institute of Biology, State University of Rio de
Janeiro, Rio de Janiero, Brazil.
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TABLE 5—Cutoff ratios in mixtures.
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* The vaginal/semen mixture gave a complete profile of the sperm
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Additional information and reprint requests:
Mark S. Nelson
North Carolina State Bureau of Investigation
Molecular Genetics Section
121 E. Tryon Rd., Raleigh, NC 27603