Development of a control region-based mtDNA SNaPshot™ selection tool, integrated into a mini amplicon sequencing...

Area of Science:

• Forensic science
• Genetics
• Molecular biology

Background:

• Mitochondrial DNA (mtDNA) analysis is crucial for forensic investigations involving degraded samples like hair and bone.
• Traditional Sanger sequencing of the mtDNA control region can be time-consuming and costly for large sample sets.
• Existing methods, such as the mini-mtDNA approach, offer robustness but can be inefficient for high-throughput screening.

Purpose of the Study:

• To develop a rapid and cost-effective screening tool for mitochondrial DNA analysis in forensic science.
• To adapt a SNaPshot-based method for the full mtDNA control region, compatible with mini-mtDNA amplicons.
• To improve the efficiency of mtDNA profiling for large-scale forensic investigations.

Main Methods:

• Development of two SNaPshot multiplex assays targeting 18 single nucleotide polymorphisms (SNPs) across the full mtDNA control region.
• Adaptation of the assays to be compatible with established mini-mtDNA amplification multiplexes.
• Selection of SNPs based on European population frequencies and validation in a Dutch population for discriminatory power.
• Inclusion of degenerate bases to mitigate allele dropout and assessment of three SNPs for haplogroup information.

Main Results:

• The developed mini-mtSNaPshot method demonstrated high discriminatory power (97.2%) in a Dutch population.
• Reliability testing confirmed no significant differences compared to traditional Sanger sequencing results.
• The screening tool is compatible with existing mini-mtDNA workflows, utilizing the same PCR products, thus conserving precious DNA extracts.

Conclusions:

• The mini-mtSNaPshot screening tool offers a forensically advantageous, rapid, and cost-effective alternative for mtDNA analysis.
• This method significantly enhances the efficiency of processing large numbers of forensic samples, particularly those with limited or degraded DNA.
• The tool provides valuable haplogroup information and maintains high reliability, making it a valuable addition to forensic genetic analysis.