A highly sensitive and nonradioactive mutation detection method based on vertical gradient temperature single-strand conformation polymorphism.
The single-strand conformation polymorphism (SSCP) method is widely used for mutation detection. The sensitivity of the method depends on several factors, most importantly on the temperature at which electrophoresis of single-stranded DNA (ssDNA) takes place. The temperature has a profound effect on the folded conformation of ssDNA. The temperature factor is predominantly determined empirically in conventional SSCP, which can be very tedious especially when a large number of different DNA samples need to be screened. We have devised a novel SSCP method based on a vertical gradient temperature (VGT), which automatically subjects ssDNA to various temperatures in the same electrophoresis. The theory behind VGT-SSCP protocol is that when ssDNA is subjected to run in a wide range of gradient temperature, it will automatically acquire optimal resolution at an optimal temperature to distinguish between the wild type and the mutant type ssDNA. The sensitivity level of mutation detection of VGT-SSCP depends on whether the corresponding optimal secondary structure of a mutant DNA strand is within the preset gradient temperature range. In summary, the VGT-SSCP is a simple and robust nonradioactive method that is more sensitive than constant-temperature SSCP in detecting unknown mutations.
Razzaghi
H
H
Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh PA, 15261 USA. razzaghi@pitt.edu
Kamboh
M I
MI
D004277:DNA, Single-Stranded / Q000032:analysis*; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity; D013696:Temperature
D004277:DNA, Single-Stranded; D008071:Lipoprotein Lipase