Improved Efficiency of Inverse PCR Mutagenesis

Generation of epitope-tagged proteins by inverse PCR mutagenesis.

The polymerase chain reaction (PCR) has been extensively used for the mutation of DNA sequences (1–5,7,8). Many variations of the protocol have been implemented, but most involve a first step in which PCR is used to modify the molecule, and a second step in which the new sequence is subcloned into a vector. A new method, named inverse PCR mutagenesis (IPCRM) has been shown to be an efficient an...

Improved inverse PCR scheme for metagenome walking.

Inverse PCR has been used for the recovery of genome regions flanking a known sequence, although its application to metagenome walking is limited due to inefficient amplification from low copy number fragments. Here we present an improved inverse PCR scheme that enables walking of rare fragments in environmental metagenomes. Our scheme includes the following steps: (i) inverse PCR in which one ...

Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully ca...

An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes.

The QuikChangeTM protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by u...

Splinkerettes--improved vectorettes for greater efficiency in PCR walking.