Touchdown Polymerase Chain Reaction (PCR).
Touchdown Polymerase Chain Reaction (PCR).
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DOI：
10.1101/pdb.prot095133
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发表时间：
2018-05-01
影响因子：
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通讯作者：
Sambrook, Joseph
中科院分区：
文献类型：
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作者：
Green, Michael R;Sambrook, Joseph
“降落聚合酶链式反应 (PCR)”是一种减少脱靶引发从而提高 PCR 特异性的方法。在降落 PCR 中，为退火步骤选择的温度最初设置为比计算的引物 Tm 高 5°C-10°C。高严格条件下的退火有利于形成完美的引物-模板杂交体。在随后的循环中，退火温度逐渐小幅降低，以便在PCR结束时，退火温度比计算的引物Tm低2°C-5°C。届时，目标序列将经历多个循环的几何扩增，因此成为 PCR 的主要产物。为了最大限度地减少 PCR 早期阶段的错误引发，降落 PCR 应始终与热启动方案结合进行。当引物的序列可能与靶标的序列不匹配时，例如，如果引物的序列是从氨基酸序列推导出来的，当模板 DNA 可能包含几个密切相关的靶标时，或者当靶标 DNA 与用于设计引物的物种不同时，则必须使用降落 PCR。
"Touchdown polymerase chain reaction (PCR)" is a method to decrease off-target priming and hence to increase the specificity of PCRs. In touchdown PCR the temperature selected for the annealing step is initially set 5°C-10°C higher than the calculated Tm of the primers. Annealing under conditions of high stringency favors the formation of perfect primer-template hybrids. In subsequent cycles, the annealing temperature is gradually decreased by a small amount so that by the end of the PCR, the annealing temperature is 2°C-5°C below the calculated Tm of the primers. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. To minimize mispriming during the early stages of the PCR, touchdown PCR should always be performed in conjunction with a hot start protocol. The use of touchdown PCR is essential when the sequence of the primer might not match that of the target-for example, if the sequence of the primer has been deduced from amino acid sequences, when the template DNA may contain several closely related targets, or when the target DNA is of a different species from that used to design the primers.