Characterization of thermostable DNA polymerase

Abstract

Methods detecting covalent lysine modifications in DNA polymerases are provided. These methods are particularly useful in determining the extent and location of a lysine modification in a DNA polymerase.

Description

Background technique [0001] Polymerase chain reaction (PCR) methods for amplifying nucleic acid sequences are well known in the art and are disclosed, for example, in US Patent Nos. 4,683,202, 4,683,195, and 4,965,188. In each cycle of PCR amplification, the double-stranded target sequence is denatured, the primers are annealed to each strand of the denatured target sequence, and the primers are extended (elongated) by the action of DNA polymerase. These steps can be summarized as denaturation, annealing and extension steps, respectively. At the high temperatures used in typical PCR, primers hybridize only to the intended target sequence. However, amplification reaction mixtures are usually assembled at room temperature, well below the hybridization temperature of the primers. Under such low stringency conditions, primers may bind only partially complementary sequences or other primers and trigger the synthesis of undesired extension products. These unwanted extension produ...

AI Technical Summary

Problems solved by technology

These unwanted extension products are amplified together with the target sequence, and this amplification competes with the amplification of the desired target sequence, reducing the efficiency of specific amplification

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