Forensic animal DNA analysis using economical two-step direct PCR

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Traditional wildlife forensic DNA analysis involves costly and time-consuming DNA sequencing.
• There is a need for more efficient and economical methods for analyzing diverse wildlife samples.

Purpose of the Study:

• To develop and validate a low-cost, two-step direct PCR assay for wildlife forensic DNA analysis.
• To assess the assay's efficiency across various mammalian sample types without prior DNA extraction.

Main Methods:

• Designed and validated a cytochrome oxidase I degenerate-universal primer pair for direct PCR.
• Tested the assay on 12 forensically relevant mammalian sample types, including bone, horn, feces, and urine.
• Compared amplification success and product quantity with commercial kits using PBS and Phire® Hot Start II DNA polymerase.

Main Results:

• Achieved an average amplification success rate of 92.5% across 350 samples.
• Obtained high-quality sequences with 100% nucleotide similarity to species databases via BLAST queries.
• Demonstrated comparable performance to commercial kits at a significantly lower cost, with assay stability up to 12 months.

Conclusions:

• The developed low-cost, two-step direct PCR assay is efficient for analyzing diverse wildlife forensic samples without DNA extraction.
• This method offers a cost-effective and time-saving alternative for wildlife forensic DNA analysis.
• The assay shows potential as a valuable tool for the forensic DNA community.