Published online 18 February 2006
Article |
Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, MD 20892-2725, USA 1Graduate Program in Biomolecular Science, University of Central Florida PO Box 162366, Orlando, FL 32816-2366, USA 2Department of Chemistry, University of Central Florida PO Box 162366, Orlando, FL 32816-2366, USA 3National Center for Forensic Science PO Box 162367, Orlando, FL 32816-2367, USA 4Laboratoire Lésions des Acides Nucléiques, LCIB-UMR-E n°3 CEA-UJF DRFMC/CEA-Grenoble, 17, avenue des Martyrs, F-38054 Grenoble Cedex 9, France
*To whom correspondence should be addressed at Building 6, Room 1A13, NICHD, NIH, 9000 Rockville Pike, Bethesda, MD 20892-2725, USA. Tel: +1 301 496 6175; Fax: +1 301 594 1135; Email: woodgate{at}nih.gov
Received December 7, 2005. Revised January 30, 2006. Accepted January 30, 2006.
For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.
DDBJ/EMBL/Gen Bank accession nos+