Elsevier

Genomics

Volume 82, Issue 3, September 2003, Pages 390-396
Genomics

Regular article
Molecular haplotype determination using allele-specific PCR and pyrosequencing technology

https://doi.org/10.1016/S0888-7543(03)00177-0Get rights and content

Abstract

Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3′ end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification.

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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