Green Michael R, Sambrook Joseph
Cold Spring Harb Protoc. 2019 Feb 1;2019(2):2019/2/pdb.prot095166. doi: 10.1101/pdb.prot095166.
The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as inverted or inside-out PCR) is used to amplify DNA sequences that flank one end of a known DNA sequence and for which no primers are available. Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites within known and flanking DNA sequences.
标准聚合酶链反应（PCR）用于扩增位于两个向内引物之间的一段DNA。相比之下，反向PCR（也称为反转或由内向外PCR）用于扩增位于已知DNA序列一端侧翼且没有可用引物的DNA序列。反向PCR涉及用限制酶消化包含已知序列及其侧翼区域的DNA制剂。各个限制片段（在总哺乳动物基因组DNA的情况下有成千上万个）通过分子内连接转化为环状，然后将环化的DNA用作PCR的模板。未知序列由两个特异性结合已知序列且方向相反的引物进行扩增。扩增反应的产物是一个线性DNA片段，其含有最初用于消化DNA的限制酶的单个位点。该位点标记了先前克隆序列与侧翼序列之间的连接处。扩增片段的大小取决于已知和侧翼DNA序列内限制位点的分布。
