Shyamala V, Ames G F
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Gene. 1989 Dec 7;84(1):1-8. doi: 10.1016/0378-1119(89)90132-7.
We have devised a strategy to extend the use of the polymerase chain reaction (PCR) to amplify double-stranded DNA when sequence information is available only at one extremity. The only information required is a short stretch of sequence used to design a gene-specific primer, which is then used in combination with a second generic vector primer at the unknown end. The primers are used in a PCR reaction after ligating the unknown end to a generic vector. Restriction, ligation, amplification and sequencing of the products can be achieved within three days. This method eliminates the laborious steps of shotgun cloning, colony screening and culturing of cells. We have used this method to take two contiguous steps beyond the histidine transport operon in Salmonella typhimurium. We also demonstrate the usefulness of this technique to do chromosome walking in the absence of any restriction data.
我们设计了一种策略，当仅在一个末端有序列信息时，可扩展聚合酶链反应（PCR）用于扩增双链DNA。唯一需要的信息是一小段用于设计基因特异性引物的序列，然后将其与未知末端的第二种通用载体引物结合使用。在将未知末端连接到通用载体后，引物用于PCR反应。产物的限制性内切酶消化、连接、扩增和测序可在三天内完成。该方法省去了鸟枪法克隆、菌落筛选和细胞培养等繁琐步骤。我们已使用此方法在鼠伤寒沙门氏菌中，在组氨酸转运操纵子之外连续推进了两步。我们还证明了在没有任何限制性数据的情况下，该技术用于染色体步移的有效性。
