Three-step PCR mutagenesis for 'linker scanning' | Zendy

X M Li | Zendy; Larry J. Shapiro | Zendy

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Three-step PCR mutagenesis for 'linker scanning'

Author(s) -

X M Li,

Larry J. Shapiro

Publication year - 1993

Publication title -

nucleic acids research

Language(s) - Uncategorized

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/21.16.3745

Subject(s) - primer (cosmetics) , biology , mutagenesis , primer dimer , genetics , site directed mutagenesis , base pair , polymerase chain reaction , microbiology and biotechnology , in silico pcr , mutation , dna , computational biology , gene , mutant , chemistry , organic chemistry , multiplex polymerase chain reaction

'Linker scanning' has been used as an efficient method for systematically surveying a segment of DNA for functional elements by mutagenesis. A three-step PCR method was developed to simplify this process. In this method, a set of 'mutation primers' was made with 6 to 8 base substitutions in the center of the primers. In the first PCR reaction, these 'mutation primers' are paired with an 3' primer from the opposite end of the analyzed sequences to form a 'ladder' of fragments containing the base pair substitutions. These are used as templates in the second PCR with the 3' primer as the only primer to generate single stranded sequences, which are used as primers in the third PCR paired with an 5' primer to complete the mutagenesis. We have tested the method in a mutation screen of the steroid sulfatase promoter. Its application to general site specific mutagenesis is discussed.

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