Highlights
- •Developmental validation of a qPCR multiplex assay, InnoQuant® HY.
- •Provides an assessment of sample quality (Degradation Index) and total and male DNA quantity.
- •Four targets are included: autosomal 80 bp, autosomal 207 bp, male 79 bp, and a 172 bp synthetic IPC.
- •Highly sensitive, reproducible, and valuable results for making informative decisions during DNA analysis process.
- •InnoQuant® HY results correlate well with downstream autosomal and Y STR DNA typing methods.
Abstract
The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets—two RE autosomal targets of
different sizes, male specific targets, and an internal positive control target—are
described herein. The ratio of the two autosomal targets provides a Degradation Index,
or a quantitative value of a sample's degradation state. The male specific targets
are multi-copy targets located on the Y chromosome, which provides information about
a sample's male DNA composition.
The experimental results demonstrate InnoQuant HY as a robust qPCR method producing
accurate DNA quantitation results even at low dynamic ranges, with reproducibility
among population groups. The system is human specific with low level higher primate
cross reactivity and is able to consistently and reproducibly detect sub-picogram
concentrations of human and human male DNA. The use of high copy number Alu and SVA
(>1000 copies per genome) retrotransposable elements as the two autosomal targets
significantly enhances both sensitivity and reproducibility of determination of DNA
quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive
multi-copy Y-chromosome specific target provides accurate quantitation of DNA from
a male in challenging male-female mixtures (i.e. sexual assault samples). Even in
the presence of a large excess of DNA from a female, accurate quantitation was achieved
with a male to female ratio of 1:128,000. Population database studies reveal an average
Short/Y target ratio of the quantification values across all four populations tested
was 1.124 ± 0.282, exhibiting the system's reproducibility across multiple populations. The results
from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about
a sample's DNA quantitation, male:female ratio, degradation state, and the presence
or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit,
a more streamlined and efficient workflow can be created that minimizes unnecessary
sample processing and retesting while maximizing recovery of probative DNA profiles
from challenging biological evidence.
Keywords
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References
- Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.Forensic Sci. Int. Genet. 2014; 13: 224-235
- Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA.Forensic Sci. Int. Genet. 2008; 3: 14-21
- Developmental validation of the Quantifiler™ Real-Time PCR Kits for the quantification of human nuclear DNA samples.J. Forensic Sci. 2005; 50: 809-825
- Developmental validation of the Quantifiler Duo DNA Quantification Kit for simultaneous quantification of total human and human male DNA and detection of PCR inhibitors in biological samples.J. Forensic Sci. 2009; 54 (correction available in Journal of Forensic Sciences. 57(2012) 282): 305-319
- A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: a diagnostic tool for STR typing.Forensic Sci. Int. Genet. 2008; 2: 108-125
Quality Assurance Standards for Forensic DNA Testing Laboratories. Available at: https://www.fbi.gov/about-us/lab/biometric-analysis/codis/qas-standards-for-forensic-dna-testing-laboratories-effective-9-1-2011 (Accessed August 11, 2016).
- Human DNA quantitation using Alu element-based polymerase chain reaction.Anal. Biochem. 2003; 315: 122-128
- Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification.Anal. Biochem. 2005; 337: 89-97
- Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.J. Forensic Sci. 2007; 52: 364-370
- Development of a real-time method to detect DNA degradation in forensic samples.J. Forensic Sci. 2012; 57: 466-471
- Forensic DNA Analysis: Current Practices and Emerging Technologies.CRC Press: Taylor & Francis Group, Boca Raton, FL United States2014 (405 pp.)
- Alu repeats and human genomic diversity.Nat. Rev. Genet. 2002; 3: 370-379
- Genome-wide analysis of the human Alu Yb lineage.Hum. Genom. 2004; 1: 167-178
SWGDAM Validation Guidelines for DNA Analysis Methods. Available at: http://www.swgdam.org/publications (Accessed January 27 2017).
ChargeSwitch gDNA Buccal Cell Kits for purification of genomic DNA from human buccal swabs. Invitrogen by Life Technologies User Guide, Revision date 12 2012.
- DNA Typing Protocols: Molecular Biology and Forensic Analysis.Eaton Publishing, Natick2000: 41-42
- Development and usage of a NIST standard reference material for real time PCR quantitation of human DNA.Forensic Sci. Int. Genet. Suppl. Ser. 2008; 1: 80-82
- SVA elements: a hominid-specific retroposon family.J. Mol. Biol. 2005; 354: 994-1007
- The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.Nature. 2003; 423: 825-837
- Tandem repeats finder: a program to analyze DNA sequences.Nucleic Acid Res. 1999; 27: 573-580
S. Sinha, Method for Genetic Detection using Interspersed Genetic Elements: a Multiplexed DNA Analysis System and Development of a Highly Sensitive Quantification System for Assessing DNA Quality in Forensic Samples. United States Patent Application Publication #20140051075A1, published on 20.02.14.
- Diversity of human copy number variation and multicopy genes.Science. 2010; 330: 641-646
- Chromosome specific DNA repeats: rapid identification in silico and validation using fluorescence in situ hybridization.Int. J. Mol. Sci. 2013; 14: 57-71
- The human Y chromosome: an evolutionary marker comes of age.Nat. Rev. Genet. 2003; 4: 598-612
- Primer3–new capabilities and interfaces.Nucleic Acids Res. 2012; 40: e115
- Enhancements and modifications of primer design program Primer3.Bioinformatics. 2007; 23: 1289-1291
- Developmental validation of the AmpFlSTR(R) Identifiler Plus PCR Amplification Kit: an established multiplex assay with improved performance.J. Forensic Sci. 2012; 57: 453-465
- Development and validation of the AmpFlSTR yfiler PCR amplification kit: a male specific, single amplification 17 Y-STR multiplex system.J. Forensic Sci. 2006; 51
- Developmental validation of the PowerPlex® Fusion System for analysis of casework and reference samples: a 24-locus multiplex for new database standards.Forensic Sci. Int. Genet. 2014; 12: 69-76
- Developmental validation of the Quantifiler HP and Trio Kits for human DNA quantification in forensic samples.Forensic Sci. Int. Genet. 2016; 21: 145-157
- Human DNA quantification and sample quality assessment: developmental validation of the PowerQuant system.Forensic Sci. Int. Genet. 2016; 23: 166-177
- LINEs and SINEs of primate evolution.Evol. Anthropol. 2010; 19: 236-249
- A Study of PCR inhibition mechanisms using real time PCR.J. Forensic Sci. 2009; 55: 25-33
- Forensic implications of PCR inhibition—A review.Forensic Sci. Int. Genet. 2012; 6: 297-305
- Comparison of dna extraction methods for multiplex polymerase chain reaction.Anal. Biochem. 2010; 398: 260-262
- Factors affecting the STR amplification success in poorly preserved bone samples.Investig. Genet. 2010; 1: 9
Article info
Publication history
Published online: April 17, 2017
Accepted:
April 14,
2017
Received in revised form:
March 26,
2017
Received:
February 2,
2017
Identification
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