Thermus thermophilus MutS, a thermostable mismatch-recognizing protein, is utilized in PCR to suppress nonspecific amplification by preventing synthesis from mismatched primers. T. thermophilus RecA also decreases nonspecific amplification by promoting proper hybridization between the primer and template. We observed that MutS and RecA function under the same reaction conditions and that MutS and RecA do not preclude each other. Furthermore, there were some DNA sequences for which only one of the 2 proteins effectively suppressed nonspecific amplification. The simultaneous use of MutS and RecA is a more attractive error-suppressing technique than the use of either of the 2 proteins alone. 1. Introduction PCR-based technologies are essential for a wide range of biosciences, ranging from basic life sciences to medical diagnoses [1–3]. The efficiency and reliability of those technologies are often decreased by nonspecific amplifications resulting from mishybridization of primers. Recently, we developed a technique to suppress nonspecific amplification by adding a thermostable mismatch-recognizing protein Thermus thermophilus MutS (ttMutS) in PCR mixture [4]. MutS is a DNA mismatch repair protein, which recognizes a variety of DNA mismatches such as base-base mismatches and base insertion loops [5, 6]. MutS is thought to bind tightly to the mismatched bases that are generated by mishybridization between primer and template to block the association of a DNA polymerase. Before we established the MutS-based error-suppressing technique, it had been reported that T. thermophilus RecA (ttRecA) protein can be used to decrease nonspecific amplification during PCR, which drastically enhances the accuracy of multiplex PCR [7]. RecA is known to be a key enzyme in homologous recombination of DNA molecules, which catalyzes pairing of 2 homologous sequences [8]. The ttRecA protein enhances the proper pairing between the primer and template to suppress nonspecific amplification. Thus, the mechanisms by which ttMutS and ttRecA suppress nonspecific amplification are quite different. In this study, we investigated the difference in the effects and properties of ttMutS and ttRecA in order to evaluate the possibility of simultaneous use of both proteins. 2. Materials and Methods 2.1. Overexpression and Purification of Proteins Thermus thermophilus MutS (ttMutS) was prepared as previously described [4]. A DNA fragment-expressing T. thermophilus RecA (ttRecA) was generated by PCR using the T. thermophilus HB8 genomic DNA as a template. The following primer pairs were used for
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