Abstract
BackgroundSite-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.ResultsThe strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.ConclusionThis strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.
Highlights
Site-directed mutagenesis is an efficient method to alter the structure and function of genes
The megaprimer is purified by gel electrophoresis and extraction before being used along with the other flanking primer in the second round of polymerase chain reaction (PCR) to generate the complete DNA sequence with the desired mutation
In this paper we describe an efficient and inexpensive single-tube two-step megaprimer-based PCR mutagenesis strategy (Fig. 1) that by-passes the cumbersome and timeconsuming gel purification step, does not require additional expensive DNA-modifying enzyme treatments, is not restrictive in primer design, and has a high mutational efficiency
Summary
Site-directed mutagenesis is an efficient method to alter the structure and function of genes. We report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. Oligonucleotide-directed mutagenesis is one of the most popular methods to introduce site-specific alterations into a DNA sequence. Various changes have been suggested [3-7] to the original strategy These megaprimer-based mutagenesis strategies are easy to use and they are very cost effective. In the first round of PCR, one of the flanking primers and the internal mutagenic primer (having desired base substitutions) are used to generate a megaprimer. The megaprimer is purified by gel electrophoresis and extraction before being used along with the other flanking primer in the second round of PCR to generate the complete DNA sequence with the desired mutation
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