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聚合酶连锁反应(polymerase chain reaction,PCR)能以酶促反应的方式使位于两段寡聚核苷酸顺序之间的DNA片段特异性扩增上百万倍。其重要用途之一就是从克隆的DNA片段中或直接从基因组DNA中制备测序模板。为了避免线状双链模板在测序反应中复性,一般使用单链DNA(ssDNA)模板。它可直接由PCR制备,也可用酶处理,电泳分离或亲和纯化等方法直接从双链DNA(dsDNA)制备。PCR与测序的结合使得扩增和测序两反应能在同一试管中进行。如果终止核苷酸或测序引物带有荧光标记,则整个程序还可望完全自动化。
Polymerase chain reaction (PCR) can catalyze the specific amplification of DNA fragments located between two oligonucleotide sequences millions of times. One of its important uses is to prepare sequencing templates from cloned DNA fragments or directly from genomic DNA. To avoid the rework of linear double-stranded templates in sequencing reactions, single-stranded DNA (ssDNA) templates are commonly used. It can be directly prepared by PCR, but also can be prepared directly from double-stranded DNA (dsDNA) by methods such as enzymatic treatment, electrophoretic separation or affinity purification. The combination of PCR and sequencing allows both amplification and sequencing reactions to proceed in the same tube. If the termination nucleotide or sequencing primer is fluorescently labeled, the whole program is expected to be fully automated.