Tag-extension-based method for sensitive and specific genotyping of single nucleotide polymorphism on microarray.
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 gamma-aminobutyric acid A receptor, beta 3 (GABAB3) gene were accurately discriminated using this method. Sanger sequencing was performed to validate these results.


CONCLUSIONS
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.
Shan
Qun
Q
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Xuzhou Normal University, Xuzhou 221116, China.
Zheng
Yuanlin
Y
Chen
Guoqing
G
Zheng
Guihong
G
Lu
Jun
J
Lv
Xiaojuan
X
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005838:Genotype; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results
D017931:DNA Primers; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides