Tag-extension-based method for sensitive and specific genotyping of single nucleotide polymorphism on microarray

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• 作者：Qun Shan ; Yuanlin Zheng ; Guoqing Chen ; Guihong Zheng ; Jun Lu ; Xiaojuan Lv
• 关键词：Single nucleotide polymorphism ; Genotyping ; Microarray ; Sensitivity ; Specificity ; Tag extension
• 刊名：Clinica Chimica Acta
• 出版年：2009
• 出版时间：3 November 2009
• 年：2009
• 卷：409
• 期：1-2
• 页码：11-17
• 全文大小：1100 K

文摘

Background

The obtainment of a large amount of single nucleotide polymorphism (SNP) information has emphasized a need for a sensitive, accurate and high-throughput strategy for SNP genotyping. We developed a reliable and potential microarray-based method to meet this demand.

Methods

A tag extension strategy is described to identify SNPs. The strategy is based on a fluorescent nucleotide extension from an extending primer that is hybridized to a bi-functional linker, which acts as an allele-specific primer that hybridizes with a PCR-amplified target DNA immobilized on a 3-dimensional (3-D) polyacrylamide gel microarray, as well as an extending template with a specific tag itself hybridized with a universal extension primer. Multiple fluorescence-dNTPs are simultaneously incorporated into the tagged linker.

Results

The method not only significantly enhanced the sensitivity, but also efficiently improved the specificity of SNP genotyping. 89 samples for 8025575C/G polymorphisms in γ-aminobutyric acid A receptor, β 3 (GABAB3) gene were accurately discriminated using this method. Sanger sequencing was performed to validate these results.

Conclusion

Our experiments successfully demonstrated that a tag-extension-based method on microarray could be used as a high-throughput and useful tool to obtain SNP information.