Rapid Forensic DNA Profiling via Real-Time Recombinase Polymerase Amplification of InDel Markers

Area of Science:

• Forensic Science
• Molecular Biology
• Biotechnology

Background:

• Traditional forensic DNA profiling methods (PCR, CE, MPS) require complex lab equipment and stable power, limiting field applications.
• There is a need for rapid, portable, and power-independent DNA typing technologies for on-site forensic identification.

Purpose of the Study:

• To demonstrate a proof-of-concept for forensic DNA typing using recombinase polymerase amplification (RPA) combined with exo probe detection.
• To evaluate the speed, sensitivity, and accuracy of this novel isothermal genotyping assay for insertion-deletion (InDel) markers.

Main Methods:

• Developed and tested an isothermal assay using RPA and allele-specific fluorescent exo probes for genotyping of eight InDel loci.
• Reactions were performed at 39 °C, combining amplification and detection within a 20-minute single step.
• Genotyping accuracy was assessed by comparing results with benchmark methods using 13 DNA samples at various input concentrations.

Main Results:

• Achieved 97.07% genotype accuracy with 1 ng DNA input compared to benchmark genotyping.
• Demonstrated reliable genotyping with DNA inputs as low as 250 pg, with partial profiles detectable at 31 pg.
• The RPA-based assay showed high sensitivity, speed (20 min), and reproducibility across multiple InDel loci.

Conclusions:

• RPA coupled with exo probe detection provides a fast, sensitive, and reproducible method for forensic InDel genotyping.
• This isothermal assay has significant potential for field-deployable forensic identification, possibly integrated into portable lab-on-a-chip platforms.
• Further optimization could lead to complete accuracy and broader application in rapid, on-site DNA analysis.