Mutagenesis polymerase chain reaction

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction. The techniques for chemical synthesis of oligonucleotides are highly developed. Very efficient automated methodologies based on solid phase synthesis are used extensively in fields that depend on the availability of defined DNA sequences.52... 

Leung, D.W., Chen, E. and Goeddel, D.V. (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1, 11-15. 

Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. 1989. Site-directed mutagenesis by overlap extension using polymerase chain reaction. Gene 77 51-59. 

List of Abbreviations GABA, gamma-aminobutyric acid type A 5HT3, 5-hydroxytryptamine type 3 SDM, site-directed mutagenesis PCR, polymerase chain reaction TRCP, targeted random chimera production SDS, sodium dodecyl sulphate... 

In 1989, a very practical mutagenesis method was described by Leung et al. 39), which was later improved by Cadwell and Joyce 40). Error-prone polymerase chain reaction (epPCR), as it is called, is based on the classical DNA amplification... 

Michael Smith and Kary B. Mullis Chemistry Oligonucleotide-directed mutagenesis and polymerase chain reaction... 

Ikeda, M., Hamano, K., and Shibata, T (1992) Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis. J. Biol Chem 267,6291-6296... 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

Fromant, M., Blanquet, S., and Plateau, P. (1995). Direct random mutagenesis of genesized DNA fragments using polymerase chain reaction. Analytical Biochemistry 224, 347-353. 

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