This study presents a multiplex typing method that couples ligase detection reaction (LDR) with repeat-polymerase chain reaction (LDR-PCR) that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA.
Study findings showed clear allele discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed. The breakdown of genomic DNA into small fragments by chemical physical factors is a challenge in the genetic identification of human remains or crime stains. One strategy that addresses some of the problems faced in analyzing degraded DNA is the reduction of polymerase chain reaction amplicon size. A number of validated mini-short tandem repeat (STR) single nucleotide polymorphism (SNP) assays have been used in casework in which current STR kits failed to achieve complete DNA profiles; however, these two techniques require an average DNA fragment length of 100-200 bp and may not be applicable to casework with extremely degraded DNA consisting of fragments less than 100 bp in length. LDR has been used to detect mutations in disease genes in clinical samples and provides an effective technique for polymorphism typing that could be applied to severely degraded DNA. In the LDR portions of the LDR-PCR technique presented, probes that are designed to be complementary to the polymorphism and to the region directly adjacent are ligated when brought into juxtaposition through hybridization with the DNA fragment. In the subsequent PCR step, the legation products are amplified using primers that are complementary to the generic priming sites built into the hybridization probes. In this study, the LDR probes were designed so that only 20-40 bp of intact DNA was required for their hybridization. 3 tables, 5 figures, and 28 references