Mismatch detection using heteroduplex analysis.
This protocol describes a technique that can be used to identify point mutations or single-base polymorphisms in heterozygous individuals. This technique takes advantage of the fact that heteroduplex molecules containing single-base mismatches can be separated under particular conditions of gel electrophoresis from nearly identical molecules containing no mismatches. RNA or DNA from a potentially heterozygous individual is amplified by PCR, and the products are denatured and allowed to renature, forming heteroduplexes. Renatured PCR products are run on nondenaturing mutation-detection-enhancement polyacrylamide gels (HydroLink MDE gels). On this gel, hybrid molecules containing a mismatch migrate more slowly than their corresponding homoduplexes. This protocol describes the analysis of unlabeled PCR products; however, radiolabeled PCR products can also be analyzed by this method.
Børresen
Anne-Lise
AL
The Norwegian Radium Hospital, Oslo, Norway.
D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods; D005826:Genetics, Medical; D020180:Heteroduplex Analysis / Q000379:methods*; D006579:Heterozygote; D006801:Humans; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide