之前的文章中，小编简介过各种进阶版的PCR（详情点这里 ），也详说了两种进阶PCR：colony PCR 和 nested PCR 。本期我们就来说说，另外一种进阶版PCR，降落式PCR （Touch-Down PCR，a.k.a. TD-PCR）。
为什么
看到Touch-Down ，就自然联想到飞机降落的过程，应该是平稳而有序，而不是忽上忽下的。那么降落式PCR就是退火温度逐循环降落的PCR。我们都知道引物的退火温度不仅仅是由引物序列决定的，不同的DNA聚合酶，不同的缓冲液都有可能影响到引物的在反应中的实际退火温度，所以根据引物序列计算出来的退火温度只是一个估测值而已。同时，我们也知道，较高的退火温度会增加引物与母板特异性结合的强度，即更有效地复制出我们想要的序列。也就是说，先使用较高的退火温度，然后退火温度持续下降直到估测值的过程，相当于是富集有效复制母板的过程。
怎么做
简单来说就两个阶段，详情请参见natural protocol [1]：
- 降温阶段。其实退火温度一般设定为（估测Tm + 10）°C，随后每完成一个循环，退火温度降低1 °C，这样持续10个循环，直到退火温度降低为估测Tm。
- 恒温阶段。退火温度保持为估测值Tm不变，继续进行20-25个循环，直到整个PCR结束。
注：在降温阶段，遵循每1-3个循环降低1-3°C即可，如果没一个循环降低一度的模式并不能给出显著的扩增结果，可以尝试不同的降温方式，但宗旨都是在10-15个循环内，将退火温度降低到估测Tm。
Extra tips
- 所有反应混合液置于冰上，直到PCR机的热循环已经启动，并且达到所需温度。
- 总PCR循环少于35为好，过多的循环总会导致各种非特异性结合。
- 如果母板GC含量过高，可以采用更长的变性时间。
Reference
[1] Nature Protocol: Touchdown PCR for increased specificity and sensitivity in PCR amplification， http://www.nature.com/nprot/journal/v3/n9/full/nprot.2008.133.html
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