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PCR induction of a TaqI restriction site at any CpG dinucleotide using two mismatched primers

ODell, SD; Humphries, SE; Day, INM; (1996) PCR induction of a TaqI restriction site at any CpG dinucleotide using two mismatched primers. GENOME RES , 6 (6) 558 - 568.

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Abstract

A final common pathway has been devised for analysis of de novo mutation at any CpG site, Artificial restriction sites can be introduced in known DNA sequences by using either or both sense and antisense mismatched PCR primers. Siting of the primers directly adjacent 5' and 3' to the CpG site yields a 52-bp PCR product, resulting from the sum of the two 25-mer oligonucleotides plus the two intervening bases (C and G), and also yields consistent digestion Fragments. Three out of four possible four-base palindromes (TaqI, HhaI, acid MspI) were investigated for mutations R329X and E80K in the human LDL receptor gene, and for mutations R395W and R612C, the Taq1 site was forced using PCR in which both primers had 3' mismatched T. Both empirically and on theoretical grounds, TaqI is the forced restriction site of choice. The approach has been adapted to the high-throughput microplate diagonal gel electrophoresis (MADGE) system for selective analysis of mutations at CpG sites, which may account for 20% of all single base variation in the human genome.

Type:Article
Title:PCR induction of a TaqI restriction site at any CpG dinucleotide using two mismatched primers
Keywords:POLYMERASE CHAIN-REACTION, POINT MUTATIONS, FAMILIAL HYPERCHOLESTEROLEMIA, ENZYMATIC AMPLIFICATION, DNA, DIAGNOSIS, GENE
UCL classification:UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Cardiovascular Science

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