Analytical Sciences

Reliable and Fast Allele-Specific Extension of 3′-LNA Modified Oligonucleotides Covalently Immobilized on a Plastic Base, Combined with Biotin-dUTP Mediated Optical Detection

Yuichi MICHIKAWA,* Kentaro FUJIMOTO,** Kenji KINOSHITA,** Seiko KAWAI,* Keisuke SUGAHARA,*^,*** Tomo SUGA,* Yoshimi OTSUKA,* Kazuhiko FUJIWARA,** Mayumi IWAKAWA,* and Takashi IMAI*

*RadGenomics Project, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan
**Bio Product Development Project Team, Sumitomo Bakelite, Ltd., 1-5-1 Murotani, Nishi, Kobe 651-2241, Japan
***Department of Oral and Maxillofacial Surgery, Tokyo Dental College, 1-2-2 Masago, Mihama, Chiba 261-8502, Japan

In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3′-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

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