Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates.
Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates.
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DOI：
10.1101/pdb.prot095141
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发表时间：
2019-02-01
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
聚合酶链反应（PCR）扩增的效率受模板DNA的核苷酸组成和序列的影响。有问题的模板包括那些具有长的均聚运行，反向重复序列或富含GC的片段-例如含有>60% G + C残基的那些-在许多哺乳动物基因的调控区中发现。富含GC残基的模板的局部区域倾向于折叠成复杂的二级结构，其在PCR循环的退火阶段期间可能不解链。此外，用于扩增富含GC的区域的引物通常具有形成自二聚体和交叉二聚体的高能力以及折叠成茎-环结构的强烈倾向，所述茎-环结构可阻碍DNA聚合酶沿着模板分子的进展。可以预见的是，全长模板DNA的扩增是低效的，并且反应产物含有高比例的较短分子，其由DNA聚合酶的阻断产生。改变引物的设计和使用热启动和降落PCR的组合有时可以提高扩增效率。更常见的是，需要多管齐下的方法，例如在扩增反应中使用增强子，调整循环方案，以及必要时设计新的引物组。该方案使用四种添加剂的混合物-甜菜碱、二硫苏糖醇（DTT）、二甲基亚砜（DMSO）和牛血清白蛋白（BSA）-用于与Taq DNA聚合酶一起使用。
The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Predictably, amplification of full-length template DNA is inefficient, and the products of the reaction contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Altering the design of the primers and using a combination of hot start and touchdown PCR can sometimes improve the efficiency of amplification. More often, a multipronged approach is required, such as the use of enhancers in the amplification reaction, adjustment of the cycling protocol, and, if necessary, designing new sets of primers. This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with Taq DNA polymerase.