首页 | 本学科首页   官方微博 | 高级检索  
     


Genotypic selection of mutated DNA sequences using mismatch cleavage analysis, a possible basis for novel mutation assays
Authors:Kortenkamp, Andreas   Jones, Chris   Baker, Jon
Affiliation:The School of Pharmacy, Department of Toxicology and of Pharmaceutical Chemistry and Biology 29–39 Brunswick Square, London WCIN 1AX. UK
Abstract:A novel technique for the selection of mutated DNA sequences,termed mismatch cleavage-polymerase chain reaction (MC-PCR),is proposed. The method is based on hybridizing genomic DNAwith a suitable probe, several 100 bp long. Mutated DNA sequenceswill form mismatched heteroduplexes which are cleaved by usingresolvases. Cleaved heteroduplexes are detected by ligationto an oligonucleotide adaptor and then amplified by using PCR.If practical, this technique would have considerable advantagesover the restriction site mutation (RSM) method. Failure toachieve cleavage efficiencies of close to 100% will not compromisesuccess. This is because positive signals (PCR amplification)arise from cleaved mutated sites and not, as in RSM, from DNAsequences resistant to cleavage by restriction endonucleases.Furthermore, the mutational target is much larger than in RSM.It would be possible to screen stretches of DNA several 100bp in length for mutations. Any mutation, independent of itslocation, could be identified. The usefulness of MC-PCR forthe genotypic selection of mutants will depend on the effectivenesswith which a small number of mismatched heteroduplexes can berecognized, cleaved and ligated. 1To whom correspondence should be addressed
Keywords:
本文献已被 Oxford 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号