Characterization of polymerase chain reaction amplification of specific alleles.
Characterization of polymerase chain reaction amplification of specific alleles.
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DOI：
10.1016/0003-2697(90)90573-r
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发表时间：
1990-04
影响因子：
2.9
通讯作者：
G. Sarkar;J. Cassady;C. Bottema;S. Sommer
中科院分区：
文献类型：
--
作者：
G. Sarkar;J. Cassady;C. Bottema;S. Sommer
在某些条件下，聚合酶链反应（PCR）可用于差异扩增一个等位基因超过另一个。为了表征这一现象，我们制作了一系列PCR引物，并确定琼脂糖凝胶电泳后是否可以检测到差异扩增。检查了两个等位基因对;一对代表颠换，一对代表转换。得出以下结论：当引物3′端或3′端倒数第二个碱基与等位基因错配时，不能检测到扩增产物，当错配距离引物3′端3个和4个碱基时，仍能观察到差异扩增，但仅在一定浓度的氯化镁下才能观察到差异扩增;（iii）错配的等位基因可以在超过40倍的匹配等位基因的存在下检测到;（iv）短至13个核苷酸的引物是有效的;和（v）扩增的特异性可能被大大增加靶DNA的浓度所压倒。
Under certain conditions, polymerase chain reaction (PCR) can be used to differentially amplify one allele over another. To characterize the phenomenon, we have made a series of PCR primers and determined whether differential amplification could be detected after agarose gel electrophoresis. Two allele pairs were examined; one pair represents a transversion and one pair represents a transition. The following conclusions emerge: (i) when the 3′ or the 3′ penultimate base of the oligonucleotide mismatched an allele, no amplification product could be detected; (ii) when the mismatches were 3 and 4 bases from the 3′ end of the primer, differential amplification was still observed, but only at certain concentrations of magnesium chloride; (iii) the mismatched allele can be detected in the presence of a 40-fold excess of the matched allele; (iv) primers as short as 13 nucleotides were effective; and (v) the specificity of the amplification could be overwhelmed by greatly increasing the concentration of target DNA.