Improved heteroduplex detection of single-base substitutions in PCR-amplified DNA.

نویسندگان

  • A V Peeters
  • M J Kotze
چکیده

Department of Human Genetics, Faculty of Medicine, University of Stellenbosch, Tygerberg, 7505, South Africa The ability to detect mutations in individuals and the subsequent DNA diagnosis of genetic diseases are becoming increasingly important for the researcher and diagnostician. Single-base substitutions (80-90%) can be detected by single-strand conformation polymorphism (SSCP) (1) or heteroduplex (2~ analysis of PCR-amplified DNA in polyacrylamide gels. Although both methods are simple, the SSCP method described earlier is used more frequently for mutation detection. (3) A drawback of this method, however, is that different mutations may require specific conditions to be detected by SSCP analysis. Furthermore, reproducibility is compromised because minor variations in specific gel running conditions can decrease the difference in mobility among conformations, thus complicating unequivocal scoring of aberrations. On the other hand, variations in the gel running conditions have little or no effect on the visualization of aberrant heteroduplex bands (A.V. Peeters, unpubl.). PCR products can be loaded directly onto a gel for heteroduplex analysis, whereas an additional denaturation step is required for SSCP analysis. In a comparative study, White et al. (4) reported detection of eight of nine mutations by heteroduplex analysis, whereas only one of these single-base mismatches could be detected by SSCP analysis. We attempted to further improve the mutation detection efficiency of the heteroduplex method by lowering the extent of cross-linking [percent crosslinking, polyacrylamide (%C)] in gels. In doing so, the resolving power and sensitivity of standard polyacrylamide gels were enhanced.

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عنوان ژورنال:
  • PCR methods and applications

دوره 4 3  شماره 

صفحات  -

تاریخ انتشار 1994