Developmental Validation of the QuantifilerTM Real-Time PCR Kits for the Quantification of Human Nuclear DNA Samples

Developmental Validation of the QuantifilerTM Real-Time PCR Kits for the Quantification of Human Nuclear DNA Samples

Quantifiler Real-Time PCR Kits are validated for quantifying human nuclear DNA in forensic samples. This study follows SWGDAM guidelines to assess the kits' performance in terms of specificity, sensitivity, and accuracy. The kits can detect as little as 32 picograms of DNA, making them suitable for low-input forensic samples. Designed for both male and total human DNA quantification, these kits enhance the efficiency of forensic DNA analysis. Ideal for forensic scientists and laboratories, this validation provides a robust framework for implementing these advanced DNA quantification methods.

Key Points

  • Validates Quantifiler Real-Time PCR Kits for human DNA quantification
  • Demonstrates high sensitivity, detecting as low as 32 picograms of DNA
  • Follows SWGDAM guidelines for forensic DNA analysis
  • Assesses specificity for human DNA and mixed samples
  • Includes performance metrics like accuracy and precision
  • newtopiccyclegrowin
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JForensicSci,July 2005, Vol. 50, No. 4
Paper ID JFS2004478
Available online at: www.astm.org
Robert L. Green,
1
B.A.; Ines C. Roinestad,
1
M.S.; Cherisse Boland,
1
B.A.; and Lori K. Hennessy,
1
Ph.D.
Developmental Validation of the Quantifiler
TM
Real-Time PCR Kits for the Quantification of
Human Nuclear DNA Samples
∗
ABSTRACT: The Quantifiler
TM
Human DNA Quantification Kit and the Quantifiler
TM
Y Human Male DNA Quantification Kit were designed for
the quantification of human genomic DNA in forensic samples. The kits use a real-time PCR-based process to quantify, respectively, total human
DNA or human male DNA only. We report the results of a developmental validation study that we performed with the Quantifiler Kits, following the
official SWGDAM guidelines. The Quantifiler Kits were tested for performance criteria such as species specificity, sensitivity, stability, precision
and accuracy, and in addition, were tested with forensic case-type samples and mixed (male:female) DNA samples. The Quantifiler Kit methods
were highly specific for human DNA, and could detect as little as 32 picograms of DNA using 2
µL of sample per assay. The accuracy and precision
of the Quantifiler Kit methods was comparable or superior to that of other quantification methods.
KEYWORDS: forensic science, DNA quantification, forensic DNA analysis, polymerase chain reaction
Molecular technologies for DNA analysis, exemplified by human
genotyping techniques using short tandem repeat (STR) sequences,
have brought about a revolution in forensic investigations. Using
these new technologies, analysts are now able to routinely work
near the ultimate limits of sensitivity and discriminatory power in
genotyping forensic casework samples. The most powerful STR
genotyping methods use the polymerase chain reaction (PCR) to
amplify target loci, allowing accurate genotype information to be
gathered from as little as picogram amounts of genomic DNA (1).
Yet, ancillary methods for the quantification of forensic DNA sam-
ples, an official requirement for STR analysis of casework samples
(2), have until recently relied on older technologies, such as slot-
blot hybridization, which were unable to match the accuracy and
sensitivity of the STR assays they were intended to precede. In addi-
tion to the limited accuracy and sensitivity of many such older DNA
quantification methods, the methods also tend to be relatively labor-
intensive and time-consuming. Often, the results of the assays are
non-objective and require a significant degree of interpretive skill
from the analyst. Although reliable and robust, most of the older
DNA quantification methods are poorly suited to high-throughput
or automated sample flow.
We report the results of developmental validation of two new
commercial kits that use “real-time” quantitative PCR to detect
and quantify human nuclear DNA, designed for use with forensic
samples. The Quantifiler
TM
Human and Quantifiler
TM
Y Human
Male DNA Quantification Kits were intended to address some of the
aforementioned limitations of older DNA quantification methods.
The high specificity of PCR amplification allows different
choices in target DNA sequences to be made, according to the
intended use of the assay. Examples of different strategies associ-
1
Applied Biosystems, Foster City, CA.
∗
A portion of the data is presented in the Quantifiler
TM
Kits User’s Manual
“Experiments and Results” section.
Received 13 Oct. 2004; and in revised form 4 Feb. 2005; accepted 5 Feb.
2005; published 25 May 2005.
ated with assay target selection are: general human-specific DNA
quantification using highly-repetitive Alu motif nuclear sequences
(3), quantification with the assessment of the degradation state of
DNA samples using the parallel amplification of differently sized
fragments of mitochondrial DNA (4), or quantification with donor
sex determination using the amplification of homologous but dis-
tinct regions of the X and Y chromosomes (4,5).
The Quantifiler kits were designed for the quantification of hu-
man nuclear DNA, and their genomic targets reflect the particular
roles for which the kits were intended. The Quantifiler Human
and Quantifiler Y Human Male kit assays detect, respectively, the
human telomerase reverse transcriptase (hTERT) locus located on
chromosome 5 (6–8), and the “sex reversal” (SRY) locus located
on the Y chromosome (9,10). The Quantifiler Human kit assay
detects total human genomic DNA (i.e., male and female), while
the Quantifiler Y Human Male kit assay detects only male DNA.
The Human kit assay is intended for general use as a preliminary
DNA quantification step to performing routine STR analysis, while
the Human Male kit assay is designed to be of use particularly
in samples with mixed male-female DNAs, such as sexual assault
evidence, where it may be useful to separately detect and quantify
male DNA from a background of female DNA. Because of the
kits’ intended use primarily as an adjunct to STR analysis, their
respective genomic target loci were chosen to be single-copy, like
the STR loci themselves, so that they would better predict the avail-
ability of amplifiable copies of the STR alleles in samples, and be
subject to the same sort of stochastic sampling effects. In addition
to the human DNA-specific elements of the Quantifiler kit reagents,
an Internal PCR Control (IPC) system—identical between the two
assays—is included as an internal quality control check confirming
the function of all system components as well as the amplifiability
of analysis samples.
The Quantifiler kit assays use “fluorogenic 5
nuclease” real-time
PCR technology for the detection of target sequence amplification
(11). This system is based on a set of two oligonucleotide PCR
primers specific for the target DNA sequence, or amplicon,plus
Copyright
C
2005 by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. 1
2 JOURNAL OF FORENSIC SCIENCES
a TaqMan
R
probe, labeled with a 5
fluorescent reporter dye and
a3
non-fluorescent quencher, that is homologous to the ampli-
con region between the PCR primers. The TaqMan probes used
in the Quantifiler kits incorporate an additional chemical modifi-
cation at their 3
ends, known as minor groove binder (MGB), to
increase the melting temperature (T
m
) and thereby allow the use of
shorter, more highly sequence-specific probes (12,13). PCR ampli-
fication is performed using an all-in-one “master mix” reagent that
includes a “hot-start” thermal-stable DNA polymerase, AmpliTaq
Gold.
R
The fluorogenic 5
nuclease assay process can be summarized
as follows. At the start of PCR thermal cycling, all TaqMan probe
molecules are intact, and the close physical proximity of the dye
and quencher suppresses the fluorescence of the dye molecules by
F
¨
orster-type energy transfer (14,15). During the course of synthe-
sizing new DNA strands from the target template, the AmpliTaq
Gold enzyme encounters the TaqMan probe annealed to the tem-
plate DNA and hydrolyzes it with its 5
→ 3
exonuclease activity.
The hydrolysis of the probe separates the dye and quencher ele-
ments, which allows the dye molecules to fluoresce more intensely.
The hydrolysis of probe molecules occurs in proportion to the am-
plification of the target sequence, and consequently, so does the re-
sulting fluorescent signal (16,17). Eventually the fluorescent signal
accumulates to the point where it can be detected over background
fluorescence, and crosses a set fluorescence threshold. The cycle
number at which the fluorescent signal crosses the threshold is de-
fined as the “Threshold Cycle”, or C
T
. There is an exact inverse
mathematical relationship between the starting copy number of tar-
get sequence molecules and the resulting C
T
, which is the basis for
quantitative PCR. In practice, the user makes a dilution series con-
taining known quantities of a human genomic DNA concentration
standard, and runs the dilutions along with samples to be quantified.
A standard curve of C
T
versus the concentration of the standard di-
lutions is automatically constructed by the analysis software, and
samples are quantified automatically by mathematical comparison
to the standard curve formula.
The Quantifiler kit reaction mix contains two independent sets
of primers and TaqMan probes, with each set constituting a “detec-
tor.” One detector uses a FAM
TM
dye-labeled TaqMan probe and
is specific for human genomic DNA (either the hTERT locus for
the Quantifiler Human kit or the SRY locus for the Quantifiler Y
Human Male kit). The other detector set uses a VIC
R
dye-labeled
TaqMan probe and, along with a synthetic DNA template added
at a fixed concentration, constitutes the IPC system. Both target
and IPC detectors are designed to amplify in parallel in every re-
action, and assessing the amplification of both systems together
allows for the positive verification of human DNA-negative sam-
ples as well as an indication of PCR inhibitors that may be present.
Figure 1 illustrates the interpretation of detector amplification. For
example, in a sample with no human-specific amplification (neg-
ative FAM dye detector result), the positive amplification of the
IPC system (positive VIC dye detector result) would verify that
all system components were functioning normally; this allows for
confirmation of true negative samples. In contrast, a sample that
showed neither amplification of the human-specific nor IPC detec-
tors would indicate the presence of PCR inhibitor in the sample,
or a failure of instrument or reagent components; in either case the
user is notified that human DNA may be present but not ampli-
fiable.
The Quantifiler kits were designed and validated for use with the
ABI PRISM
R
7000 and ABI PRISM 7900HT Sequence Detection
Systems (SDS) instruments, which includes the SDS software that
controls the instruments and automatically performs data analysis.
FIG. 1—Quantifiler Human kit assay amplification plots for positive,
negative and inhibited reactions. Amplification plots for the human detec-
tor (FAM
TM
reporter dye) and IPC detector (VIC
R
reporter dye) are indi-
cated, showing reaction fluorescence (Rn) versus cycle number. A positive
result is defined when a detector’s amplification plot crosses the fluores-
cence threshold line, and the threshold cycle, or C
T
, is set at that cycle
number. Human and IPC detector results can be used to confirm a human-
negative assay result or to detect assay failure due to the presence of PCR
inhibitor in the analysis sample or faulty assay setup. A: Human DNA-
positive reaction results, with positive amplification of both human and
IPC detectors; B: Human DNA-negative reaction results, with negative
amplification of the human detector but positive (confirmatory) amplifica-
tion of the IPC detector; C: Assay results for a sample with total PCR
inhibition, where both human and IPC detectors were negative.
Developmental validation of the Quantifiler kits was performed
according to the guidelines of the Scientific Working Group on
DNA Analysis Methods (SWGDAM) (18). Although these guide-
lines were developed mainly with STR genotyping assays in mind,
we sought to apply them as closely as possible to the Quantifiler kit
validation since they are intended to be used as part of the same pro-
cess flow that includes the STR assays themselves. However, cer-
tain of the guideline criteria, such as those concerning measurement
GREEN ET AL.
r
QUANTIFILER VALIDATION 3
standards for alleles or the mode of inheritance of genetic mark-
ers, were not applicable to these quantification assays and were not
tested.
Materials and Methods
Sources and Preparation of Genomic DNAs Used in Testing
Human genomic DNAs were obtained from different commer-
cial suppliers or were extracted from single-source and casework
samples by a forensic testing laboratory using an organic (phe-
nol:chloroform) purification procedure (19). A set of 50 human ge-
nomic DNA samples, most from individuals of African-American
ethnicity (approximately half male, half female), referred to in this
paper as the “resolution panel,” were extracted from whole blood
specimens by the supplier (SeraCare Life Sciences, Inc., Oceanside,
CA). A set of 450 genomic DNAs known as the DNA Polymor-
phism Discovery Resource (referred to as the “Coriell panel” in
this paper), was prepared by the provider (Coriell Cell Reposito-
ries, Camden, NJ, catalog number MPDR450). The Coriell panel,
made up of approximately half male and half female individu-
als, represented multiple ethnicities and geographic regions of the
United States; it consisted of the following number of individuals
of different ethnic groups: 120 European-American, 120 African-
American, 60 Mexican-American, 30 Native American and 120
Asian-American (20). Additional individual DNAs were obtained
from the following suppliers: Raji cell line DNA (male), Lofs-
trand Labs Limited, Gaithersberg, MD; 9948 cell line DNA (male),
Marligen Biosciences, Inc., Ijamsville, MD; “007” DNA extracted
from whole blood (male), Serological Research Institute, Rich-
mond, CA; K-562 cell line DNA (female), American Type Culture
Collection, Rockville, MD. All human DNA samples were geno-
typed using the AmpFSTR
R
Identifiler
R
PCR Amplification Kit
(Applied Biosystems), which allowed independent verification of
the sex of each donor by assessing the alleles of the amelogenin
locus.
A panel of DNAs from primate species was obtained from BIOS
Laboratories, Inc., New Haven, CT; the sex of the donor animals
was not known for these samples. Additional DNAs from individual
animals of known sex were prepared from whole blood specimens
using an ABI PRISM
TM
6100 Nucleic Acid PrepStation procedure,
or were provided by the Oregon State Fish and Game Laboratory.
Multiple genomic DNA samples of bacterial and yeast species were
prepared from pure cultures using the IsoQuick
TM
Nucleic Acid
Extraction Kit, Orca Research Inc., Bothell, WA; these purified
DNA samples were combined in several pools, each containing
DNA from five individual species.
DNA Quantification Using UV Absorbance, Dye Intercalation
and Slot Blot Hybridization Methods
Different DNA quantification methods were used to assess the ac-
curacy of the Quantifiler kit assays. Spectrophotometric absorbance
at 260 nm wavelength (A
260
method) was done with a Lambda 10
BIO spectrophotometer (PerkinElmer Instruments, Shelton, CT).
The DNA concentrations of samples were then calculated using a
standard conversion formula (21):
Conc.(
µg/mL) = 50 × A
260
.
The PicoGreen
R
dsDNA Quantitation Kit (Molecular Probes,
Eugene, OR), referred to herein as the dye intercalation assay, was
performed according to the microplate protocol. Fluorescence emis-
sion was measured on an ABI PRISM 7000 Sequence Detection
System instrument. After subtracting the background fluorescence
of a blank sample from all standards and unknown samples as di-
rected in the kit protocol, the standard curve data points were plotted
on a graph and a regression line with associated linear regression
formula was determined using Microsoft Excel
R
2000. Each sam-
ple was then quantified by entering its background-subtracted nu-
merical fluorescence value into the regression formula and solving
for DNA concentration.
Slot blot hybridization DNA quantification assays were per-
formed using the QuantiBlot
R
Human DNA Quantitation Kit (Ap-
plied Biosystems) according to the protocol for chemiluminescent
autoradiography. Quantification was done by visually comparing
the blot intensity of samples against those of a two-fold dilution se-
ries of a human genomic DNA standard. In some instances, analysis
samples were diluted so as not to exceed the maximum concentra-
tion range of the QuantiBlot kit assay.
Quantifiler Human and Quantifiler Y DNA Quantification
Kit Assays
The Quantifiler kit assays were performed according to the proto-
col specified in the User’s Manual. Quantification standard dilutions
were first made by serially diluting the 200 ng/
µL stock solution
from the kits to the following concentrations: 50, 16.7, 5.56, 1.85,
0.62, 0.21, 0.068 and 0.023 ng/
µLinT
10
E
0.1
Buffer (10 mM Tris-Cl
pH 8.0, 0.1 mM Na
2
EDTA) that contained 20 µg/mL of molecular
biology grade glycogen as a stabilizer (Roche Applied Science, In-
dianapolis, IN). Master mix was formulated by combining 10.5
µL
of Primer Mix (specific for the Quantifiler Human or Quantifiler
Y kits) and 12.5
µL of Quantifiler PCR Reaction Mix per reaction,
multiplied by the number of reactions required, then dispensing the
mixintoamABIPRISM
TM
96-Well Optical Reaction Plate (Ap-
plied Biosystems) at 23
µL per reaction. 2.0 µL sample volume was
added per reaction, with duplicate reactions of each quantification
standard and single reactions of each analysis sample being run.
After all reactions were set up, the optical plate was then covered
with a clear plastic Optical Adhesive Cover (Applied Biosystems),
pressed into place with a plastic applicator tool, and covered with
a foam compression pad. The plate was then placed in the 96-well
sample block of an ABI PRISM 7000 or ABI PRISM 7900HT
Sequence Detection System (SDS) instrument. The SDS software
controlling the 7000 or 7900HT instruments was programmed for
the following thermal cycle:
95
◦
C/10 min hold → 40 cycles of[95
◦
C/15 sec, 60
◦
C/60 sec]
After themal cycling was completed (approximately 1:45 hours),
data analysis was performed by the SDS software to generate stan-
dard curve data for quantification standards and quantification re-
sults for unknown samples.
AmpFSTR Identifiler PCR Amplification Kit Genotyping Assays
STR analysis was performed using the standard protocol for the
AmpFSTR Identifiler PCR Amplification Kit assay. Identifiler kit
reactions were analyzed on an ABI PRISM 3100 Genetic Ana-
lyzer with Data Collection Software v1.1. Electropherogram data
was analyzed with GeneScan
R
Software v3.7.1 and Genotyper
R
Software v3.7 for use with the Windows
R
NT operating system.
Species Specificity Studies
The specificity of the Quantifiler Human and Quantifiler Y as-
says was assessed for a large number of human DNA samples and
for panels of genomic DNA from non-human vertebrate species
/ 17
End of Document
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FAQs of Developmental Validation of the QuantifilerTM Real-Time PCR Kits for the Quantification of Human Nuclear DNA Samples

What are the main findings of the Quantifiler validation study?
The validation study confirms that the Quantifiler Real-Time PCR Kits are highly effective for quantifying human nuclear DNA in forensic samples. The kits demonstrated the ability to detect low quantities of DNA, specifically as little as 32 picograms, which is crucial for forensic applications. The study also established the kits' specificity for human DNA, ensuring reliable results even in mixed samples. Overall, the findings support the kits' integration into forensic laboratories for improved DNA analysis efficiency.
How does the Quantifiler kit improve forensic DNA analysis?
The Quantifiler kit enhances forensic DNA analysis by providing a highly sensitive and specific method for quantifying human DNA. Its ability to detect low DNA concentrations allows forensic scientists to work with challenging samples that may contain minimal DNA. Additionally, the kit's design facilitates the differentiation between male and female DNA, which is particularly useful in cases involving mixed samples. By streamlining the quantification process, the Quantifiler kit contributes to more efficient and accurate forensic investigations.
What guidelines were followed in the Quantifiler validation?
The validation of the Quantifiler Real-Time PCR Kits adhered to the guidelines set forth by the Scientific Working Group on DNA Analysis Methods (SWGDAM). These guidelines provide a framework for assessing the performance of DNA analysis methods, ensuring that the kits meet established standards for specificity, sensitivity, and accuracy. By following these protocols, the study ensures that the Quantifiler kits are reliable tools for forensic DNA quantification, thus supporting their use in real-world applications.
What types of DNA samples were tested with the Quantifiler kits?
The Quantifiler kits were tested with a variety of DNA samples, including those extracted from whole blood and forensic casework samples. The study included both male and female DNA, allowing for comprehensive validation of the kits' performance in mixed DNA scenarios. Additionally, the kits were evaluated using DNA from different ethnic backgrounds to ensure their broad applicability in forensic contexts. This diverse testing approach enhances the reliability of the Quantifiler kits across various forensic applications.

Related of Developmental Validation of the QuantifilerTM Real-Time PCR Kits for the Quantification of Human Nuclear DNA Samples