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Tools for Improving the Quality of Aged, Degraded, Damaged, or Otherwise Compromised DNA Evidence

NCJ Number
237840
Date Published
Author(s)
John R. Battista
Annotation
This study’s primary objective was to develop tools that will facilitate STR DNA genotyping by improving the quality of the DNA found in degraded forensic samples and enhancing the ability to retrieve amplifiable DNA from forensic samples.
Abstract
The study succeeded in developing an in-vitro method that uses three purified proteins in enabling the selective capture of specific DNA sequences. This method relies on the use of a targeting DNA fragment, a sequence that is complementary to the sequence the examiner wishes to retrieve. Combining this fragment, the three proteins, and appropriate cofactors allows recovery of homologous DNA from solution with high efficiency. This process, referred to as in-vitro double-strand break repair reaction, requires the following three proteins: RecA protein, single-strand binding (SSB) protein, and a DNA polymerase. To date, all three proteins are obtained from Escherichia coli. RecA and SSB are needed for the identification of the region of homology and promoting invasion by the 3’ end of single strand DNA molecule recombining with duplex DNA. The DNA polymerase extends the 3’ ends, sealing the spliced fragments formed by the action of RecA and SSB. This reaction can be used to repair double strand breaks found between the STR and the primer sites for STR amplification. If the targeting fragment includes at its 5’ end a feature that allows efficient retrieval such as biotin that can be harvested with streptavidin-coated magnetic beads, recombined fragments can be isolated and concentrated for further manipulation. In order to establish that in-vitro double strand break repair is possible, an assay was developed that used the plasmid pUC19 cleaved by a pair of restriction enzymes. 6 figures, 2 tables, and 26 references
Date Created: February 27, 2012