DNA POLYMERASES WITH IMPROVED REVERSE TRANSCRIPTASE ACTIVITY

There exists a need for enzymes with improved reverse transcriptase activity to increase the sensitivity of diagnostic assays which use RNA targets. In our lab we have produced a collection of chimeric mutant DNA polymerases with modulated proofreading activity. A mutagenized library of one of these DNA pols was screened for improved extension rates on a primed M13 template. Some of the mutants displayed the ability to perform improved RT‐PCR using either Mn2+ or Mg2+. One of the DNA pol mutations, D640G, was transferred to the parent DNA pols of the chimera, Thermatoga maritima and Thermus sp. Z05. These mutants also demonstrated the ability to perform efficient RT‐PCR with reduced RT incubation times. In Z05 the D580 position was mutated to all 19 other amino acids and the resulting proteins were purified. The D580G and K mutants were found to have improved RT activity and increased RT‐PCR sensitivity using TaqMan‐based detection. Enzymatic characterization revealed a 12.5‐fold improvement in the catalytic efficiency of Mn2+‐activated RT for Z05 D580G over wild‐type. The Z05 D580K enzyme also showed significant activity when Mn2+ was replaced by Mg2+ allowing for higher fidelity RT‐PCR.