Double Strand Break Repair of Highly Degraded DNA

DNA extracted from biological stains is often intractable to analysis. This may be due to a number of factors including a low copy number of starting molecules, the presence of soluble inhibitors or damaged DNA templates. Previous work in this laboratory has shown that double strand and single strand breaks are significant contributors to the non-typeability of damaged DNA templates extracted from forensic samples that are often exposed to environmental insults). This proposal seeks to repair double strand breaks, restoring sufficient genomic integrity to permit DNA typing using a panel of single nucleotide polymorphism (SNP) loci. Three methods will be attempted. The first is a simple gap filling prior to strand denaturation during the DNA amplification process. A modification of this requires the addition of repair substrates that are complementary to the sequences flanking a SNP on both DNA strands, providing a matrix for repair polymerization and facilitating the recovery of amplifiable fragments. Two more complex methods, based upon in vivo cellular double strand break repair processes, will also be developed and tested.

ca/ncf