Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA

doi:10.1016/j.fsigen.2008.07.004

Forensic Science International: Genetics

Volume 3, Issue 1, December 2008, Pages 14-21

https://doi.org/10.1016/j.fsigen.2008.07.004 Get rights and content

Abstract

Multiplex human short tandem repeat analysis demands reliable DNA quantification to consistently produce interpretable genotypes. The Plexor^® HY System is a multiplex quantitative PCR assay to quantify total human and male DNA. We performed developmental validation of the Plexor^® HY System to demonstrate the performance capabilities and limitations of the assay for forensic applications. Validation studies examined: (a) human specificity, (b) sensitivity, (c) quantification of degraded DNA, (d) impact of inhibitors, (e) male/female mixture and Y-assay male specificity, (f) reproducibility and concordance and (g) population studies.

Introduction

Multiplex short tandem repeat (STR) analysis is the core technology in DNA-based human identification. These assays require a defined range of template quantities to produce optimal results. In addition to accurate sample quantification, assessment of sample quality and sensitive detection are necessary to determine how best to proceed with sample analysis.

Quantitative PCR (qPCR) has displaced hybridization-based methods for human-specific DNA quantification in forensic applications. qPCR has reduced the rate of false-negative STR results due to lack of sensitivity and increased the objectivity of data interpretation by providing a numerical output rather than requiring a visual comparison of band intensities. However, some current qPCR methods do not allow simultaneous quantification of total human and male DNA or do not have a level of sensitivity that consistently exceeds that of subsequent STR assays.

The Plexor^® HY System is a qPCR assay that simultaneously quantifies total human DNA and male DNA [1], [2], [3] using the Plexor^® technology, which results in decreasing fluorescence as the amplification progresses [4], [5], [6], [7]. The triplex configuration allows co-amplification of a human autosomal sequence, a human Y-chromosomal sequence and a novel exogenous control sequence to quantify total human DNA and male DNA and provide an internal PCR control (IPC), respectively.

The autosomal primers are labeled with fluorescein and amplify a 99 bp sequence from the human RNU2 locus. The RNU2 locus encodes a small nuclear RNA involved in pre-mRNA processing. This region is conserved among primates and organized as a tandemly repeated motif (∼6 kb each) on the long arm of chromosome 17 [8], [9], [10], [11], [12], [13], [14], [15]. The Y-chromosome primers are labeled with CAL Fluor^® Orange 560 (Biosearch Technologies, Inc.) and target a 133 bp sequence from the testis-specific protein, Y-encoded (TSPY) locus. The TSPY gene is involved in spermatogenesis and is conserved in primates [15], [16], [17], [18]. The TSPY locus is within the DYZ5 region, a 20 kb repeated motif on the Y chromosome. The IPC primers are labeled with CAL Fluor^® Red 610 (Biosearch Technologies, Inc.) and detect a novel IPC sequence, which is included as a template in all reactions. The amplified IPC product is 150 bp. Data from the IPC amplifications are used to monitor amplification inhibition. A fourth dye, IC5, is included in all wells and used as a passive reference. Data from the three amplifications can be normalized to the passive reference signal to reduce the impact of instrument-specific signal fluctuation.

The findings presented here document the basic performance characteristics of the Plexor^® HY System as part of a manufacturer's validation. Given these results, laboratories implementing the Plexor^® HY System may consider omitting some of these studies from their internal validation as previously described by the manufacturer [19].

Section snippets

DNA

The Plexor^® HY Male Genomic DNA Standard, provided with the Plexor^® HY System, was used to generate all standard curves. This DNA is a mixture of several human male DNAs and is not derived from cell lines. Except where noted, we generated standard curves by amplifying a fivefold serial dilution of the DNA standard from 16 pg/μl to 50 ng/μl. We purified male and female human DNA for use as unknown samples from liquid blood using organic extraction [20] or from buccal swabs. Buccal swabs were

Species specificity

DNA purified from forensic samples commonly contains a mixture of human DNA and contaminating DNA from bacteria, fungi, viruses or other organisms, and some samples may not include any human biological material. Consequently, STR genotyping assays for forensic use are generally reactive to human and primate DNA only [22], [23]. Similarly, the quantification system must not react to non-primate DNA.

Six primate, 23 non-primate mammal, two non-mammalian animal, two yeast, six prokaryotic and two

Conclusions

Quantification of both total human and male DNA in complex forensic samples provides critical information on how to proceed with sample analysis. Quantification must consistently suggest an appropriate amount of sample to produce interpretable STR results. This information must be generated with minimal sample consumption and confidently identify samples with inadequate amounts of DNA for STR analysis. Quantification must not be significantly affected by the presence of contaminating DNA, and

Acknowledgement

The authors would like to thank Terri Sundquist (Promega Corporation) for assistance in preparation and review of this manuscript.

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¹: Current address: Third Wave Technologies, 502 South Rosa Road, Madison, WI 53719, United States.

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Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA

Abstract

Introduction

Section snippets

DNA

Species specificity

Conclusions

Acknowledgement

J. Mol. Biol.

J. Mol. Biol.

Genomics

Genomics

Forensic Sci. Int.

Enzymatic repair of an expanded genetic information system

Nucleic Acids Res.

A third base pair for the polymerase chain reaction: inserting isoC and isoG

Nucleic Acids Res.

Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence

J. Am. Chem. Soc.