NuMY-A qPCR Assay Simultaneously Targeting Human Autosomal, Y-Chromosomal, and Mitochondrial DNA.

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Accurate DNA quantification is crucial for forensic analysis, especially with limited or degraded samples.
• Existing methods for nuclear DNA quantification are available, but tools for simultaneous nuclear and mitochondrial DNA quantification are scarce.
• Mitochondrial DNA quantification is vital for samples with low nuclear DNA content and for optimizing next-generation sequencing assays.

Purpose of the Study:

• To introduce NuMY, a novel quantitative real-time PCR (qPCR) method for parallel quantification of human nuclear and mitochondrial DNA.
• To assess the male DNA proportion in mixtures and detect PCR inhibition.
• To provide a reliable tool for forensic DNA analysis, adhering to MIQE guidelines.

Main Methods:

• Development of a new quantitative real-time PCR (qPCR) assay named NuMY.
• Parallel quantification of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA).
• Inclusion of targets for autosomal DNA, male-specific DNA, and mtDNA, along with inhibition controls.

Main Results:

• NuMY demonstrated comparable nDNA quantification results to existing techniques for high-quality DNA extracts.
• The method proved reliable for challenging forensic samples, including mixtures, inhibited samples, and degraded DNA.
• NuMY successfully performed parallel quantification of nDNA and mtDNA, assessed male DNA proportions, and detected PCR inhibition.

Conclusions:

• NuMY is a valuable tool for accurate and comprehensive DNA quantification in forensic science.
• The method supports informed decisions regarding downstream genotyping and optimizes the use of limited forensic samples.
• NuMY addresses the need for simultaneous nDNA and mtDNA quantification, particularly for complex and low-quantity samples.