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COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a modified Polymerase Chain Reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA. The ability to preferentially amplify and identify minority alleles and low-level somatic DNA mutations in the presence of excess wildtype alleles is useful for the detection of mutations. Detection of mutations is important in the case of early cancer detection from tissue biopsies and body fluids such as blood plasma or serum, assessment of residual disease after surgery or chemotherapy, disease staging and molecular profiling for prognosis or tailoring therapy to individual patients, and monitoring of therapy outcome and cancer remission or relapse. Common PCR will amplify both the major (wildtype) and minor (mutant) alleles with the same efficiency, occluding the ability to easily detect the presence of low-level mutations. The capacity to detect a mutation in a mixture of variant/wildtype DNA is valuable because this mixture of variant DNAs can occur when provided with a heterogeneous sample – as is often the case with cancer biopsies. Currently, traditional PCR is used in tandem with a number of different downstream assays for genotyping or the detection of somatic mutations. These can include the use of amplified DNA for RFLP analysis, MALDI-TOF (matrix-assisted laser-desorption–time-of-flight) genotyping, or direct sequencing for detection of mutations by Sanger sequencing or pyrosequencing. Replacing traditional PCR with COLD-PCR for these downstream assays will increase the reliability in detecting mutations from mixed samples, including tumors and body fluids. ==COLD-PCR Method Overview== The underlying principle of COLD-PCR is that single nucleotide mismatches will slightly alter the melting temperature (Tm) of the double-stranded DNA. Depending on the sequence context and position of the mismatch, Tm changes of 0.2-1.5 °C (0.36-2.7 °F). are common for sequences up to 200bp or higher. Knowing this the authors of the protocol took advantage of two observations: *Each double-stranded DNA has a ‘critical temperature’ (Tc) lower than its Tm. The PCR amplification efficiency drops measurably below the Tc. *The Tc is dependent on DNA sequence. Two template DNA fragments differing by only one or two nucleotide mismatches will have different amplification efficiencies if the denaturation step of PCR is set to the Tc. Keeping these principles in mind the authors developed the following general protocol: #Denaturation stage. DNA is denatured at a high temperatureusually . #Intermediate annealing stage. Set an intermediate annealing temperature that allows hybridization of mutant and wildtype allele DNA to one another. Because the mutant allele DNA forms the minority of DNA in the mixture they will be more likely to form mismatch heteroduplex DNA with the wildtype DNA. #Melting stage. These heteroduplexes will more readily melt at lower temperatures. Hence they are selectively denatured at the Tc. #Primer annealing stage. The homo-duplex DNA will preferentially remain double stranded and not be available for primer annealing. #Extension stage. The DNA polymerase will extend complementary to the template DNA. Since the heteroduplex DNA is used as template, a larger proportion of minor variant DNA will be amplified and be available for subsequent rounds of PCR. There are two forms of COLD-PCR that have been developed to date. Full COLD-PCR and Fast COLD-PCR. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「COLD-PCR」の詳細全文を読む スポンサード リンク
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