Highlights
- •A qPCR assay is described which provides an assessment of sample quality.
- •The multiplex is a TaqMan assay comprised of three fluorescently labeled probes.
- •Three targets are studied: autosomal 80 bp, autosomal 207 bp, and a synthetic IPC.
- •Developmental validation studies are described.
- •InnoQuant™ proves to be a useful tool for forensic DNA testing laboratories.
Abstract
There is a constant need in forensic casework laboratories for an improved way to
increase the first-pass success rate of forensic samples. The recent advances in mini
STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet
few tools are available that can simultaneously provide an assessment of quantity,
inhibition, and degradation in a sample prior to genotyping. Currently there are several
different approaches used for fluorescence-based quantification assays which provide
a measure of quantity and inhibition. However, a system which can also assess the
extent of degradation in a forensic sample will be a useful tool for DNA analysts.
Possessing this information prior to genotyping will allow an analyst to more informatively
make downstream decisions for the successful typing of a forensic sample without unnecessarily
consuming DNA extract. Real-time PCR provides a reliable method for determining the
amount and quality of amplifiable DNA in a biological sample.
Alu are Short Interspersed Elements (SINE), approximately 300 bp insertions which are distributed throughout the human genome in large copy number.
The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to
a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human
genome, which minimizes the individual specific variation possible when using a multi-copy
target quantification system. This study utilizes two independent retrotransposon
genomic targets to obtain quantification of an 80 bp “short” DNA fragment and a 207 bp “long” DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™.
The ratio of the two quantitation values provides a “Degradation Index”, or a qualitative
measure of a sample's extent of degradation. The Degradation Index was found to be
predictive of the observed loss of STR markers and alleles as degradation increases.
Use of a synthetic target as an internal positive control (IPC) provides an additional
assessment for the presence of PCR inhibitors in the test sample.
In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that
accurately predicts the status of a biological sample, will be a valuable tool for
deciding which DNA test kit to utilize and how much target DNA to use, when processing
compromised forensic samples for DNA testing.
Keywords
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Article info
Publication history
Published online: August 18, 2014
Accepted:
August 11,
2014
Received in revised form:
May 27,
2014
Received:
February 13,
2014
Identification
Copyright
© 2014 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.
