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Mismatched PCR
A method for detection of a point mutation that neither produces nor destroys a restriction site; intended especially for screening large numbers of individuals. One of the PCR primers is designed so that it is complementary to the sequence immediately upstream (or downstream) from the mutation, and near its 5'-end (or 3'-end) contains a mismatched base. The mismatch and the nearby mutated base (but not the normal base) when amplified generate a new restriction site. The amplified mutant DNA (unlike the amplified normal DNA), when digested with appropriate endonucleases, electrophoresed and probed with an appropriate labelled oligonucleotide, will reveal a new, smaller restriction fragment. Alternatively, the mismatched primer may be designed to create a new restriction site with the normal, but not the mutant, DNA.
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- Industry/Domain: Biology
- Category: Biochemistry; Molecular biology
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