聚合酶链反应
化学
DNA
聚合酶链反应优化
聚合酶
反聚合酶链反应
多重位移放大
模板
聚合酶
DNA聚合酶
分子生物学
多重聚合酶链反应
硅胶PCR
底漆二聚体
热启动PCR
PCR的应用
生物
重组酶聚合酶扩增
基因
生物化学
DNA提取
水热
纳米技术
材料科学
作者
Michael R. Green,Joseph Sambrook
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2019-02-01
卷期号:2019 (2): pdb.prot095141-pdb.prot095141
被引量:15
标识
DOI:10.1101/pdb.prot095141
摘要
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.
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