title: Detecting single base substitutions as heteroduplex polymorphisms.
abstract: We have developed a sensitive technique for detecting single base substitutions in polymerase chain reaction (PCR) products from individuals heterozygous for polymorphisms or new mutations. This technique takes advantage of the formation of heteroduplexes in the PCR between different alleles from heterozygous individuals. These heteroduplexes can be detected on polyacrylamide gels because they migrate slower than their corresponding homoduplexes. Using PCR, we have generated a series of point mutations in a defined region of DNA in the equine infectious anemia virus (EIAV). Each mutation is the result of a single base substitution. By mixing the PCR products amplified from these mutations with one another, as well as with wildtype PCR products, we can generate heteroduplexes in which the identity of the mismatched bases is known. We detected eight of nine point mutations using this technique. We have also modified the electrophoretic conditions to optimize the detection of these heteroduplexes. In addition, the usefulness of this technique is demonstrated by its ability to detect a mutation in the cystic fibrosis gene that is the result of a single base substitution. This technique should prove useful for rapidly screening large numbers of individuals for new mutations or polymorphisms.
authors.affiliation: National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201.
authors.forename: M B
authors.initials: MB
authors.lastname: White
authors.forename: M
authors.initials: M
authors.lastname: Carvalho
authors.forename: D
authors.initials: D
authors.lastname: Derse
authors.forename: S J
authors.initials: SJ
authors.lastname: O'Brien
authors.forename: M
authors.initials: M
authors.lastname: Dean
mesh_terms: D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D015342:DNA Probes; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
chemical_list: D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes; D004247:DNA