A Multiplex PCR-Ligase Detection Reaction Assay for Human Identity Testing

Abstract

Current methods of forensic DNA identification at our facilities use PCR for typing single nucleotide and tandem repeat polymorphisms. Unfortunately, these PCR-based methods are relatively expensive and time consuming and are not well suited for total automation. The ligase detection reaction (LDR) when used in conjunction with PCR offers distinct advantages. In LDR, two adjacent primers hybridize to the target and are ligated only when there is perfect complementarity at the junction. Since Taq DNA ligase used in LDR is thermostable, several rounds of thermal cycling may be used to unambiguously distinguish any single nucleotide polymorphisms. We have developed a coupled multiplex PCR-LDR assay to type single base variations at 12 biallelic loci, giving a power of discrimination of 1.12 × 10⁵. The 12 loci are PCR amplified in a single reaction using a unique two-step method that produces similar amounts of multiplexed products without the need to carefully adjust primer concentrations or PCR conditions. Following PCR, these products are used in a single LDR to generate products that are resolved and typed on an Applied Biosystems 373 DNA sequencer, creating an LDR profile. Our ability to easily generate similar amounts of product in a 12-locus multiplex PCR amplification is the basis for expanding the assay to type 30 biallelic loci to give a theoretical power to discriminate one individual in 10¹².