Kirchhoff F, Desrosiers R C
New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102.
PCR Methods Appl. 1993 May;2(4):301-4. doi: 10.1101/gr.2.4.301.
Oligonucleotide primers corresponding to variable region 3 (V3) of simian immunodeficiency virus (SIV) were randomly mutagenized during synthesis by doping each of the four nucleoside phosphoramidites with a small amount of the other three. PCR was then used to incorporate the altered sequences into larger, clonable DNA fragments by spliced overlap extension (SOE). With the composition of the phosphoramidites used, 53 of the 100 clones analyzed were unique, having one or more point mutation within the 84-bp target sequence. These 53 unique clones contained an average of 2.1 nucleotide substitutions and 1.5 amino acid substitutions per clone within the target V3 sequence. Of the internal 25 amino acid positions within the V3 domain, 23 were changed at least once. This method should be generally useful for the construction of libraries of random point mutations within a defined target DNA sequence.
在合成过程中，通过在四种核苷亚磷酰胺中分别掺入少量其他三种，对与猿猴免疫缺陷病毒（SIV）可变区3（V3）对应的寡核苷酸引物进行随机诱变。然后通过拼接重叠延伸（SOE），利用聚合酶链式反应（PCR）将改变后的序列整合到更大的、可克隆的DNA片段中。在所使用的亚磷酰胺组成下，分析的100个克隆中有53个是独特的，在84个碱基对的靶序列内有一个或多个点突变。这些53个独特的克隆在靶V3序列内每个克隆平均含有2.1个核苷酸替换和1.5个氨基酸替换。在V3结构域内的25个内部氨基酸位置中，有23个至少改变过一次。该方法对于在定义的靶DNA序列内构建随机点突变文库通常应是有用的。
