A method for specific cloning and sequencing of human hprt cDNA for mutation analysis.
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.
Simpson
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Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709.
Crosby
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Skopek
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D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008214:Lymphocytes; D009154:Mutation; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification
D012333:RNA, Messenger; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase