Novel Thermostable Y-Family Polymerases: Applications for the PCR Amplification of Damaged or Ancient DNAs

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 simplify damaged DNA, thereby restricting its usefulness in forensic applications. This article reports the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygioiobus azoricus, and Sulfurisphaera ohwkuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. By using a blend of Taq and Dpo4-like enzymes, the authors obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. This research involves the first steps toward using a Y-family polymerase to replicate the damaged DNA template, so as to generate one or more lesion-free copies of the template that could be subsequently amplified by a higher fidelity PCR enzyme. Descriptions of materials and methods address the identification and cloning of novel Dpo4-like genes, the construction of chimeric enzymes, the purification of Dpo4-like enzymes, in vitro primer extension assays, PCRs on undamaged DNA, Genomic DNA sample preparation, and PCR amplification and detection of Alu sequences. 6 figures and 28 references