A 26plex autosomal STR assay to aid human identity testing*.

Area of Science:

• Molecular Biology
• Forensic Genetics
• Genomics

Background:

• Short tandem repeat (STR) analysis is a cornerstone of forensic DNA profiling.
• Developing multiplex assays with a high number of loci is crucial for increasing discrimination power and efficiency.
• Previous multiplex assays faced challenges in multiplexing numerous loci within a single reaction.

Purpose of the Study:

• To develop and validate a novel multiplex assay capable of amplifying 26 distinct STR loci simultaneously.
• To optimize primer design and reaction conditions for a 26-locus assay utilizing five-dye chemistry.
• To assess the performance of the multiplex assay across a broad range of genomic DNA concentrations.

Main Methods:

• Design of primers for 25 autosomal STR loci and the amelogenin (AMEL) sex-typing locus.
• Development of a multiplex PCR strategy with amplicons ranging from 65 to 400 base pairs (bp).
• Utilization of a five-dye chemistry system, with one dye designated for the sizing standard.
• Testing the assay's sensitivity with genomic DNA samples ranging from 2 ng down to 100 pg, using 30 PCR cycles.

Main Results:

• Successful development of a single-tube multiplex assay amplifying 26 STR loci (25 autosomal + AMEL).
• Amplicons were strategically distributed across a 65-400 bp range, compatible with standard capillary electrophoresis systems.
• The assay demonstrated robust amplification from a wide DNA input range (2 ng to 100 pg) within 30 PCR cycles.
• The five-dye chemistry, including a dedicated sizing standard dye, facilitated accurate fragment sizing.

Conclusions:

• The developed 26-locus STR multiplex assay is a powerful tool for forensic applications.
• This assay offers high efficiency and discrimination for reference sample testing.
• It holds significant potential for improving the analysis of complex relationship evaluations in forensic investigations.