Regular articleMolecular haplotype determination using allele-specific PCR and pyrosequencing technology
Section snippets
Specificity of AS-PCR
Initially, we investigated the difference in PCR specificity between complementary primers and primers carrying a 3′-end mismatch. To this end, allele-specific forward primers for both allelic variants of the SNP at position −20 of the β2-adrenergic receptor gene were designed (Fig. 1, Fig. 2). Two DNA samples, T/T and C/C, respectively at the SNP position, were chosen for an AS-PCR experiment in which an annealing temperature gradient ranging from 40 to 73°C was tested for matched and
Discussion
Pyrosequencing technology offers a rapid, reliable, and highly quantitative method for evaluation of SNPs occurring on a DNA strand specifically amplified by AS-PCR. The quantitative measurements obtained in a pyrogram provide a great advantage since, first, the efficiency of PCR amplification can be judged from the peak height, and second, any amplification of the nonspecific allele would be detected as a portion of a peak. The technology also enables an efficient analysis, in that SNPs
Model gene and DNA samples
The human β2-adrenergic receptor gene was used as a model system for the development of a method for molecular haplotyping. Thirteen SNPs organized into 12 haplotypes within a 1.5-kb sequence have been previously documented in this gene. The SNPs at positions −1023, −468, −20, and +79 (numbering according to [6]) were analyzed on DNA samples prepared from human blood by QIAamp columns (Qiagen GmbH, Germany).
PCR amplification
For evaluation of the AS-PCR, fragments that could be used directly for genotyping were
Acknowledgements
We thank Lars Berg (Pyrosequencing AB) for linguistic revision of the manuscript.
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