Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.
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).
Michikawa
Yuichi
Y
RadGenomics Project, National Institute of Radiological Sciences, Chiba, Japan. y_michi@nirs.go.jp
Fujimoto
Kentaro
K
Kinoshita
Kenji
K
Kawai
Seiko
S
Sugahara
Keisuke
K
Suga
Tomo
T
Otsuka
Yoshimi
Y
Fujiwara
Kazuhiko
K
Iwakawa
Mayumi
M
Imai
Takashi
T
D000469:Alkaline Phosphatase; D000483:Alleles; D001710:Biotin; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics; D005838:Genotype; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019809:Streptavidin; D013482:Superoxide Dismutase / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000235:genetics; D000099156:Superoxide Dismutase 2
D000469:Alkaline Phosphatase; D001710:Biotin; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D009711:Nucleotides; D009841:Oligonucleotides; D019809:Streptavidin; D013482:Superoxide Dismutase; D053773:Transforming Growth Factor beta1; D000099156:Superoxide Dismutase 2; C085108:APEX1 protein, human