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Vol. 9, Issue 1, 72-78, January 1999

METHODS
Single-Tube Genotyping without Oligonucleotide Probes

Søren Germer, and Russell Higuchi1

Roche Molecular Systems (RMS), Alameda, California 94510 USA

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.

1   Corresponding author.

9:72-78 ©1999 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/99 $5.00
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