Polymerase chain reaction site directed mutagenesis

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... 

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... 

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... 

In 1993 the Nobel Prize was awarded for two techniques which rely heavily on the ability to synthesise oligodeoxynucleotides, namely site directed mutagenesis and the polymerase chain reaction. 

Kary B. Mullis (1944- ), United States. For his invention of the polymerase chain reaction (PCR) method. Michael Smith (1932-2000), Canada. For his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies. ... 

Ho, S.N., Hunt, H.D. et. al. (1989) Site directed Mutagenesis by Overlap Extension Using the Polymerase Chain Reaction, Gene, 77 51-59. 

Reikofski, J. and Tao, B.Y. (1992) Polymerase chain reaction (PCR) techniques for site-directed mutagenesis. Biotechnol, Adv, 10, 535-547. 

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