Double Strand Break Repair of Highly Damaged DNA

• NCJ Number: 236690
• Author(s): Jack Ballantyne Ph.D.
• Date Published: October 2009
• Length: 98 pages
• Grant Number(s): 2006-DN-BX-K005
• Agencies: NIJ-Sponsored
• Publication Type: Report (Study/Research)

Annotation

This project attempted to repair "in the test tube" highly fragmented genomic DNA in order to retrieve a DNA profile from otherwise intractable environmentally compromised samples.

Abstract

After attempting various procedures that used current technology, researchers concluded that such technology cannot reconstitute two contiguous fragments of DNA to bring it back to its native state after environmental conditions have compromised the samples. The efforts determined that the sequence complexity of genomic DNA poses an insurmountable computation barrier to reconstituting two contiguous fragments of DNA back to the native state, which is required for the successful repair of double strand breaks. Two general strategies were used in an attempt to repair a double strand break. The "molecular biology" approach involved capturing repair substrates that composed oligonucleotides that subtended a SNP locus of interest and whose function was to capture the appropriate genomic fragments from the degraded sample. The successful repair of oligonucleotide substrates that mimicked some SNP loci was obtained by using capture oligonucleotides; however, all subsequent studies that involved fragmented genomic DNA failed to show any signs of repair. A second strategy used the "biochemical" approach, which recapitulated the cell's biochemical machinery for double-strand break repair in the test tube, specifically non-homologous end joining (NHEJ). NHEJ also yielded successful repair of oligonucleotide substrates that mimicked some SNP loci; however, all subsequent studies that involved fragmented genomic DNA failed to show any signs of successful NHEJ repair. An alternative but related biochemical pathway, MMEJ (Microhomology-Mediated End-Joining Pathway), was reconstituted, but also failed to repair genomic DNA. Instead of direct repair, researchers also developed and tested a modified oligonucleotide ligation assay (OLA) that was designed to recover profiles from fragmented DNA without actually repairing the DNA. This approach also failed. 23 figures, 5 tables, 30 references, reviewers' comments, and a listing of presentations in which project results were presented