Detection of known mutation by proof-reading PCR.
Proof-reading PCR (PR-PCR) is designed to detect known mutations within genomic DNA. It differs from standard PCR approaches in that one of the two primers has its 3' end aligned with a putative mutation site, and has its 3'-OH replaced by a blocking group. Distinguishing a mutant gene from wild-type depends upon preferential removal of the blocked 3' terminal nucleotide by the polymerase proof-reading activity when it is mismatched with the template. Preferential removal of the blocked nucleotide allows subsequent extension and selective amplification, and provides the basis for distinguishing mutant from normal genes. This method has been used here to detect a transition mutation within the P53 gene of HaCaT cells with verification by direct sequencing of the selectively amplified DNA.
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Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati, College of Medicine, PO Box 670521, Cincinnati, OH 45267-0521, USA.
Stambrook
P J
PJ
D002294:Carcinoma, Squamous Cell; D004252:DNA Mutational Analysis / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis* / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured
D009692:Nucleic Acid Heteroduplexes; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase