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Single-tube genotyping without oligonucleotide probes.
- Source :
- Genome Research; January 1999, Vol. 9 Issue: 1 p72-78, 7p
- Publication Year :
- 1999
-
Abstract
- We report the development of a self-contained (homogeneous), single-tube assay for the genotyping of single-nucleotide polymorphisms (SNPs), which does not rely on fluorescent oligonucleotide probes. The method, which we call Tm-shift genotyping, combines allele-specific PCR with the discrimination between amplification products by their melting temperatures (Tm). Two distinct forward primers, each of which contains a 3'-terminal base that corresponds to one of the two SNP allelic variants, are combined with a common reverse primer in a single-tube reaction. A GC-tail is attached to one of the forward allele-specific primers to increase the Tm of the amplification product from the corresponding allele. PCR amplification, Tm analysis, and allele determination of genomic template DNA are carried out on a fluorescence-detecting thermocycler with a dye that fluoresces when bound to dsDNA. We demonstrate the accuracy and reliability of Tm-shift genotyping on 100 samples typed for two SNPs, and recommend it both as a simple and inexpensive diagnostic tool for genotyping medically relevant SNPs and as a high-throughput SNP genotyping method for gene mapping.
Details
- Language :
- English
- ISSN :
- 10889051 and 15495469
- Volume :
- 9
- Issue :
- 1
- Database :
- Supplemental Index
- Journal :
- Genome Research
- Publication Type :
- Periodical
- Accession number :
- ejs19036266