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Addressing Quality and Quantity: The Role of DNA Repair and Whole Genome Amplification in Forensically Relevant Samples

NCJ Number
249511
Author(s)
Bruce Budowle
Date Published
May 2013
Length
86 pages
Annotation
This study explored methods for damaging DNA in its native complexed form prior to the testing of the PreCR Repair Mix (New England BioLabs) in an attempt to repair damaged template DNA prior to its use in PCR.
Abstract
The generation of significantly damaged DNA samples was challenging, requiring extensive time and substantial effort. The conditions are described in this report so that other researchers may be assisted in producing sufficiently damaged DNA for repair studies. Repair efforts with the PreCR Repair Mix were performed on DNA from environmentally damaged bloodstains, human skeletal remains, and bleach-damaged whole blood. Although the Pre CR Repair protocol improved the performance of STR profiling of bleach-damaged DNA and, to a lesser extent, environmentally damaged DNA, the results were varied and unreliable. A modified PreCR protocol outperformed the manufacturer-recommended approach, but still produced inconsistent results and only nominal increases in allele peak heights. The report advises that PreCR should not be considered for DNA repairs due to its poor performance. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. The original DOP-PCR primer was modified by removing the unnecessary restriction site and reducing the required bases on the 3 end of the primer. This allowed for an overall more robust amplification of shorter fragments from damaged samples, contemporary skeletal samples, and Civil-War-era bone compared with results obtained by standard DNA typing and the unmodified DOP-PCR method. The new DOP-PCR primers show promise for WGA of degraded DNA. 29 tables, 23 figures, 60 references, and listings of products disseminated and researchers and collaborators