Chester N, Marshak D R
W.M. Keck Structural Biology Laboratory, Cold Spring Harbor, New York 11724.
Anal Biochem. 1993 Mar;209(2):284-90. doi: 10.1006/abio.1993.1121.
We report a method for optimizing the specificity of product formation in the polymerase chain reaction (PCR). This technique is based on the use of dimethyl sulfoxide (DMSO) and takes into account primer Tm. The reduction in Tm by DMSO is directly correlated with renaturation temperature such that a DMSO gradient reflects a temperature gradient. We use this relationship to show that optimum product formation usually occurs at or within several degrees of the midpoint Tm of a given primer pair. We illustrate these correlations using three examples deriving PCR products from a human cDNA library, representing the casein kinase II alpha and beta subunits as well as the 5' untranslated region for the beta subunit. By following product formation as a function of renaturation temperature, we postulate rules for cycle design based on primer Tm. Implications for the use of degenerate primers are discussed.
我们报告了一种优化聚合酶链反应（PCR）中产物形成特异性的方法。该技术基于二甲基亚砜（DMSO）的使用，并考虑了引物的熔解温度（Tm）。DMSO引起的Tm降低与复性温度直接相关，因此DMSO梯度反映了温度梯度。我们利用这种关系表明，最佳产物形成通常发生在给定引物对的中点Tm或其几度范围内。我们用三个从人cDNA文库中获得PCR产物的例子来说明这些相关性，这些例子分别代表酪蛋白激酶II的α和β亚基以及β亚基的5'非翻译区。通过跟踪作为复性温度函数的产物形成情况，我们基于引物Tm提出了循环设计规则。还讨论了简并引物的使用意义。
