Efficient Long-PCR Site-Specific Mutagenesis of a High GC Template

An efficient and optimized PCR method with high fidelity for site-directed mutagenesis.

We have developed an efficient method for site-directed mutagenesis using two subsequential rounds of PCR. In this method, PCR conditions are optimized to favor high fidelity of Taq DNA polymerase in the presence of equimolar concentrations of MgCI2 and dNTP in the reaction mixture (pH 5.5-6.2). This method makes use of a pair of universal primers and the multiple cloning site of pUC/M13 vector...

Highly Efficient Site-Specific Mutagenesis in Malaria Mosquitoes Using CRISPR.

Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Ano...

Rapid and efficient site-specific mutagenesis without phenotypic selection.

Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The appro...

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in compleme...

Protocol of a Seamless Recombination with Specific Selection Cassette in PCR-Based Site-Directed Mutagenesis