Fast multiplexed polymerase chain reaction for conventional and microfluidic short tandem repeat analysis

Area of Science:

• Forensic Science
• Molecular Biology
• Biotechnology

Background:

• Short tandem repeat (STR) amplification time is influenced by thermal cycler ramp rates, reaction mix components, and vessel properties.
• Previous multiplex amplifications using microfluidic biochips and conventional tubes took 17.3 and 19 minutes, respectively.

Purpose of the Study:

• To optimize and evaluate a fast multiplex PCR protocol for STR analysis.
• To assess the performance of the optimized protocol across a range of DNA template concentrations.
• To explore the potential for a fully integrated microfluidic forensic DNA analysis system.

Main Methods:

• Optimized a 28-cycle amplification protocol for multiplex STR analysis.
• Utilized microfluidic biochip-based and conventional tube-based thermal cyclers.
• Characterized PCR performance using DNA template levels from 0.006 to 4 ng.
• Separated and detected amplified products on a microfluidic electrophoresis system (Genebench-FX).
• Generated CODIS-compatible profiles using Profiler Plus ID, COfiler, and Identifiler primer sets.

Main Results:

• Achieved multiplex amplifications in as little as 17.3 minutes.
• Generated alleles with signal strengths above calling thresholds.
• Demonstrated heterozygous peak height ratios greater than 0.65.
• Observed incomplete nontemplate nucleotide addition and stutter below 15%.
• Confirmed full CODIS-compatible profiles were generated.

Conclusions:

• The fast multiplex PCR approach significantly reduces process time and cost for STR analysis.
• This method enables the development of a fully integrated microfluidic forensic DNA analysis system.
• The optimized protocol is robust across a wide range of DNA template inputs.