Preferential PCR amplification of alleles: mechanisms and solutions.
Preferential PCR amplification of alleles: mechanisms and solutions.
复制标题
DOI：
10.1101/gr.1.4.241
复制
发表时间：
1992-05-01
期刊：
影响因子：
--
通讯作者：
Higuchi, R
中科院分区：
文献类型：
--
作者：
Walsh, P S;Erlich, H A;Higuchi, R
杂合子样本中一个等位基因相对于另一个等位基因的优先PCR扩增可能会导致该样本的基因分型不正确或不明确。有几种机制可能会潜在地导致这种优先的PCR扩增。首先，如果反应的条件(变性温度(Tden)、在Tden‘盐中的时间和共溶剂浓度等)，等位基因之间显著的GC%差异可以导致优先扩增。允许一个等位基因变性，但不允许另一个等位基因变性(差异变性)。例如，人类白细胞抗原-DQA基因座的Dqa1.1、-1.2和-1.3等位基因不能在89摄氏度下扩增；这些相同的条件仍然允许Dqa2、-3和-4等位基因的扩增。然而，当TDEN设置在推荐的TDEN为94摄氏度时，即使在多达102个循环的扩增后，不同的HLA-DQA等位基因之间的扩增效率也没有发现差异。第二，在基于聚合酶链式反应的基因分型系统中，来自不同等位基因的聚合酶链式反应产物的长度不同，可以优先扩增较短的等位基因产物。在不同条件下扩增可变数目串联重复序列标记D17S5(YNZ22)的实验表明，当Taq聚合酶为限制性时，较小的等位基因产物优先被扩增。如果目标DNA被充分降解，也可以发生VNTR等位基因的优先扩增。第三，当样本的初始基因组数量非常少时，每个等位基因拷贝数的随机波动可能会导致似乎是优先扩增。最后，由于引物和特定的等位基因模板之间的不匹配，一个等位基因的DNA合成的启动效率可能低于另一个等位基因，从而导致另一个等位基因的优先扩增。讨论了避免优先放大的一般策略。
The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences between alleles if the conditions of the reaction (denaturation temperature (Tden), duration at the Tden' salt and co-solvent concentrations, etc.) allow the denaturation of one allele but not the other (differential denaturation). For example, the DQa1.1, -1.2, and -1.3 alleles of the HLA-DQa locus do not amplify at a Tden < 89 degrees C; these same conditions still allow amplification of the DQa2, -3, and -4 alleles. However, no differences in amplification efficiency were found between the different HLA-DQa alleles when the Tden was set at the recommended Tden of 94 degrees C, even after as many as 102 cycles of amplification. Second, for PCR-based genetic typing systems in which the PCR products from different alleles differ in length, preferential amplification of the shorter allelic product can occur. Experiments in which the variable number tandem repeat (VNTR) marker D17S5 (YNZ22) was amplified under various conditions suggest that the smaller allelic products are amplified preferentially when Taq polymerase is limiting. Preferential amplification of VNTR alleles can also occur if the target DNA is sufficiently degraded. Third, when the initial number of genomes sampled is very small, stochastic fluctuation in the number of copies of each allele can result in what appears to be preferential amplification. Finally, less efficient priming of DNA synthesis of one allele versus another can occur because of mismatches between the primer and the specific allelic template, resulting in preferential amplification of the other allele. General strategies to avoid preferential amplification are discussed.