Site-directed mutagenesis using a double-stranded DNA fragment as a PCR primer

Site-directed mutagenesis using a double-stranded DNA fragment as a PCR primer.

The polymerase chain reaction (PCR) (1) has found wide application in identifying gene mutations in patients with genetic diseases. It is often useful to test the effect of specific mutations on gene expression in vitro. We describe a PCR protocol to rapidly and efficiently introduce specific mutations found in patient material into cloned DNA using double-stranded PCR fragments containing the ...

Rapid PCR method for site-directed mutagenesis on double-stranded plasmid DNA.

Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion.

DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact. Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion. A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containi...

A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA.

A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two se...

A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase.

A novel method for site-directed mutagenesis of DNA sequences based on the use of the PCR is described. The method uses two oligonucleotide primers that contain the desired sequence change and overlap at their 5' ends. In addition, the thymine residues in the overlap region have been substituted with deoxyuracil. Amplification of the template plasmid by PCR results in incorporation of the prime...