Detecting single base substitutions as heteroduplex polymorphisms
| Autor: | Marga Belle White, Magda Carvalho, Michael Dean, David Derse, Stephen J. O'Brien |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Male
Cystic Fibrosis DNA Mutational Analysis Molecular Sequence Data Polymerase Chain Reaction law.invention Equine infectious anemia chemistry.chemical_compound law Genetics Humans Allele Polymerase chain reaction COLD-PCR Polymorphism Genetic Base Sequence biology Point mutation Nucleic Acid Heteroduplexes Wild type DNA biology.organism_classification Molecular biology Pedigree chemistry Female DNA Probes Heteroduplex |
| Zdroj: | Genomics. 12:301-306 |
| ISSN: | 0888-7543 |
| DOI: | 10.1016/0888-7543(92)90377-5 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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