Simpson D, Crosby R M, Skopek T R
Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709.
Biochem Biophys Res Commun. 1988 Feb 29;151(1):487-92. doi: 10.1016/0006-291x(88)90619-5.
A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesis and subsequent amplification of hprt sequences is accomplished using Thermus aquaticus DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5' ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both in vitro and in vivo.
已开发出一种用于特异性扩增和克隆人hprt cDNA的方法，该方法可用于突变序列分析。从TK6淋巴母细胞中分离信使RNA，并用于以寡聚dT为引物通过逆转录酶产生第一链cDNA。在聚合酶链反应（PCR）过程中，使用嗜热栖热菌DNA聚合酶和hprt特异性引物完成hprt序列的第二链合成及后续扩增。在PCR引物的5'端构建了方便的限制性酶切位点，以便将hprt片段克隆到M13mp19中。使用PCR反应产物或克隆到M13mp19中的片段作为底物，可用特异性引物对hprt进行双脱氧测序。这种通用的克隆/测序方法可用于分析在体外和体内获得的人细胞中的hprt突变。
