Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia
| Autor: | Dan Y. Wu, R. B. Wallace, L. Ugozzoli, B. K. Pal |
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| Rok vydání: | 1989 |
| Předmět: |
Genetics
Multidisciplinary biology Inverse polymerase chain reaction Gene Amplification Multiple displacement amplification Anemia Sickle Cell Molecular biology Globins law.invention genomic DNA law Multiplex polymerase chain reaction Allele-specific oligonucleotide biology.protein Humans Primer (molecular biology) Oligonucleotide Probes Alleles Polymerase chain reaction Polymerase Research Article Fluorescent Dyes |
| Zdroj: | Proceedings of the National Academy of Sciences. 86:2757-2760 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.86.8.2757 |
| Popis: | A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing. |
| Databáze: | OpenAIRE |
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