| 摘要 |
A specific and sensitive method for the identification of mutations in a gene. The method comprises the steps of: obtaining nucleic acids from a sample, preparing from said nucleic acids large segments of said gene of at least about 700bp each. The next step involves independent digestion of each of said segments with at least two different restriction enzymes, each digestion performed in separate reaction. Each of the chosen restriction enzyme/s has a single restriction site in said segment and this site is different from the restriction site/s of the other restriction enzyme/s. Digestion according to the present invention should give for each of said segments two unequal fragments of different length each, for each restriction enzyme. The digestion fragments obtained in the former step are then subjected to SSCP analysis, whereby said two unequal fragments of different length each are separated. Finally, the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions of the same segment, that were separated on different lanes in the SSCP analysis, are compared to a control wild-type sample, and any difference in the resulting gel-banding patterns of overlapping fragments, between the tested sample and the control, indicates the existence of mutation in said segment. |
