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- W1945286931 abstract "In combination with PCR amplification, restriction enzyme analysis forms a rapid and general technique to identify gene lesions that change a restriction enzyme site. After the PCR product has been digested with a known restriction enzyme and subjected to electrophoresis, a specific pattern of bands appears on the gel. More than 100 different restriction enzyme recognition sequences are known, but because only one in six of the basepairs in the human haploid genome are covered by a naturally occurring restriction enzyme recognition site (1), >80% of mutations are not directly detectable. In such instances, appropriate restriction sites can be artificially created by siting a single nucleotide mismatch in the 3′ end of an oligonucleotide primer immediately adjacent to the mutation site (2). After incorporation of the primer into a PCR product, the (usually common) variant nucleotide of one allele matches the sequence of the primer to introduce a restriction enzyme recognition site, and the corresponding nucleotide of the other allele does not. After digestion of the PCR product, cleavage will occur completely, partially, or not at all, and this identifies which alleles were present in the template DNA. A limitation of the method is that a restriction site must be created within the primer in the confines of the genomic DNA sequence; for some mutations, there may not be a potential restriction site to engineer.We have adapted this approach to detect rare mutations at mutation-prone CpG sites (3). Artificial restriction sites can be established in known DNA sequences by using either sense or antisense (or both) PCR primers to force the site through mismatch of the 3′-terminal base with the target. In each case, forcing or not, both primers can be sited directly adjacent to the target CpG dinucleotide, enabling a general basis for identifying mutations at this mutation-prone …" @default.
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- W1945286931 date "1998-01-01" @default.
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- W1945286931 title "CpG-PCR Combined with Sample Pooling and Mutant Enrichment for CpG Mutation Screening in Population Studies" @default.
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- W1945286931 doi "https://doi.org/10.1093/clinchem/44.1.183" @default.
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