An integrated system of ABO typing and multiplex STR testing for forensic DNA analysis

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Traditional forensic DNA analysis often requires separate tests for blood group and STR profiling.
• There is a need for integrated systems to improve efficiency and reduce sample requirements in forensic investigations.

Purpose of the Study:

• To develop and validate a single-reaction multiplex amplification system for simultaneous ABO and autosomal short tandem repeat (STR) genotyping.
• To assess the system's performance, sensitivity, and robustness for forensic applications.

Main Methods:

• Designed primers for simultaneous amplification of 15 autosomal STR loci (including CODIS, Penta D, Penta E) and six ABO genotypes, plus Amelogenin for sex determination.
• Optimized polymerase chain reaction (PCR) conditions, including reagent concentrations, cycle numbers, and annealing temperatures.
• Validated the system using 364 bloodstain samples and 32 casework samples, assessing sensitivity, mixture detection, and performance with degraded DNA.

Main Results:

• The system successfully detects ABO genotypes and 15 STR loci in a single reaction, with amplicons ranging from 75bp to 500bp across four fluorescent dyes.
• Optimal DNA template range is 250pg to 2ng, with a low detection limit of 125pg (63pg for ABO loci).
• Demonstrated robust performance with minor allele detection in 1:9 mixtures (83%), tolerance to degraded DNA, and successful validation on real forensic samples.

Conclusions:

• The developed multiplex system provides both ABO blood group and comprehensive STR profiles from a single reaction, enhancing forensic analysis efficiency.
• The system meets Chinese National Standards and SWGDAM guidelines, proving to be a valuable tool for forensic DNA typing and identification.