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NCJRS Abstract

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NCJ Number: 228502 Find in a Library
Title: Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone
Journal: Journal of Forensic Sciences  Volume:54  Issue:5  Dated:September 2009  Pages:1001-1007
Author(s): Kenneth D. Eilert, M.S.; David R. Foran, Ph.D.
Date Published: September 2009
Page Count: 7
Type: Report (Study/Research)
Format: Article
Language: English
Country: United States of America
Annotation: This study examined 10 thermostable polymerases from 6 bacterial species for their ability to amplify DNA in the presence of bone-derived or individual PCR inhibitors.
Abstract: Of the enzymes tested, Tfl, Tfi, Tgo, and Pfx50 were most susceptible to skeletal inhibitors, with Tfl inhibited even by fresh bone; thus, none appeared to be good candidates for skeletal DNA analyses. Hot-start Taq variants performed better in the presence of skeletal inhibitor than did standard Taq, with Ex Taq HS leading over AmfliTaq Gold and HotMaster Taq. Tth consistently outperformed all other polymerases in high concentrations of skeletal PCR inhibitors; it, along with Ex Taq HS, appeared most advantageous for assaying skeletal remains. BSA improved amplification success for almost all of the enzymes in the presence of the skeletal inhibitors and is a useful adjuvant in general. Therefore, polymerases that are prepackaged with BSA in their buffers may be more suitable for overcoming skeletally derived inhibitors compared with products that lack the adjuvant Buffer BSA levels, however, are not necessarily optimal for use on skeletal DNAs; thus, addition of BSA to at least 400ng/ml should be considered for maximal inhibitor relief. Two ancient human bones that exhibited high levels of PCR inhibition in previous analyses (38) were selected to be a source of inhibitors: a tibia fragment from Diaporit, Albania, dated fifth-seventh century AD; and a thoracic vertebra fragment from Butrint, Albania, dated fifth-seventh century A.D. Nonhuman control DNA was obtained from a fresh porcine ox coxae fragment. DNA extraction was performed in a Labconco Purifier PCR Enclosure. In addition to describing material processing and DNA Extraction, this report describes assayed thermostable polymerases, bone inhibitor PCR assays, individual inhibitor PCR assays, and optimizing PCR parameters. 2 tables, 1 figure, and 52 references
Main Term(s): Criminology
Index Term(s): Bone analysis; Death investigations; DNA fingerprinting; Forensic sciences; Investigative techniques
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