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

The polymerase chain reaction (PCR) 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...

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Bibliographic Details
Published inNucleic acids research Vol. 18; no. 16; p. 4947
Main Authors Giebel, L B, Spritz, R A
Format Journal Article
LanguageEnglish
Published England 25.08.1990
Subjects
Base Sequence
DNA Mutational Analysis
Gene Amplification
Gene Expression Regulation
Humans
Monophenol Monooxygenase - genetics
Monophenol Monooxygenase - metabolism
Oligodeoxyribonucleotides
Polymerase Chain Reaction
Templates, Genetic
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Summary:The polymerase chain reaction (PCR) 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 mutations of interest. The procedure uses a double-stranded PCR product amplified from an M13 clone containing a mutation as one primer and an oligonucleotide derived from another exon of the gene as a second primer to generate a PCR product that can then be easily cloned into the desired vector. We identified two mutations in exon 4 of the tyrosinase gene in patients with tyrosinase-negative oculocutaneous albinism.
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ISSN:0305-1048
1362-4962
DOI:10.1093/nar/18.16.4947