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ligase chain reaction


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A technique that allows a specific region of a DNA molecule to be screened for mutations. The targeted region is denatured and reannealed to a set of four oligonucleotides. For each strand of the target DNA, two complementary oligonucleotides are designed, which will base pair in tandem with the strand, leaving only a gap where the right end of one molecule abuts the left end of the other. This gap can be sealed by a DNA ligase, and subsequently the complete strand can serve as a substrate against which new pairs of nucleotides can anneal and be ligated. As one cycle follows another, the ligated pairs of oligonucleotides are successively amplified. A thermal cycler is used to alternatively heat and cool the reaction mixture to allow first separation and then binding of complementary molecules. A thermostabile ligase isolated from Thermus aquaticus (q.v.) serves as the sealing agent. One can now test other DNAs to see if they contain base sequence variations in the targeted region. Any mutation that interfers with the base pairing of the oligonucleotides when they bind to the targeted sequence will prevent the ligase from joining the right and left ends of these two molecules. Therefore no amplification will take place. So the failure of amplification shows that the test DNA contains nucleotide sequences that do not match oligonucleotide sequence sites critical for ligation. The LCR therefore provides a rapid, accurate way for screening the patients suffering from a genetic disease for mutant alleles of a targeted gene. See Chronology, 1990, Barany.

Subjects: Genetics and Genomics.


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