Chimeric Thermostable DNA Polymerases with Reverse Transcriptase and Attenuated 3‘−5‘ Exonuclease Activity

DNA聚合酶 聚合酶 校对 分子生物学 核酸外切酶 克莱诺碎片 化学 DNA聚合酶Ⅱ DNA聚合酶Ⅰ 初级 DNA 逆转录酶 过程性 底漆(化妆品) 生物化学 DNA钳 生物 DNA聚合酶δ 聚合酶链反应 基因 有机化学
作者
Nancy J. Schönbrunner,Ellen H. Fiss,Olga Budker,Susanne Stoffel,Christopher L. Sigua,David H. Gelfand,Thomas W. Myers
出处
期刊:Biochemistry [American Chemical Society]
卷期号:45 (42): 12786-12795 被引量:22
标识
DOI:10.1021/bi0609117
摘要

The synthesis of accurate, full-length cDNA from low-abundance RNA and the subsequent PCR amplification under conditions which provide amplicon that contains minimal mutations remain a difficult molecular biological process. Many of the challenges associated with performing sensitive, long RT/PCR have been alleviated by using a mixture of DNA polymerases. These mixtures have typically contained a DNA polymerase devoid of 3'−5' exonuclease, or "proofreading", activity blended with a small amount of an Archaea DNA polymerase possessing 3'−5' exonuclease activity, since reverse transcriptases lack 3'−5' exonuclease activity and generally have low fidelity. To create a DNA polymerase with efficient reverse transcriptase and 3'−5' exonuclease activity, a family of mutant DNA polymerases with a range of attenuated 3'−5' exonuclease activities was constructed from a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These "designer" DNA polymerases were fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues resulted in proteins in which DNA polymerase activity was unaffected, while proofreading activity ranged from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicated that the mutations affected catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5−15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR. The utility of these enzymes was evaluated in a RT/PCR assay to generate a 1.7 kb amplicon from HIV-1 RNA.
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