Sequencing-by-Ligation Using Oligonucleotide Probes with 3′-Thio-Deoxyinosine
We have developed a novel sequencing-by-ligation (SBL) system employing oligonucleotide probes containing 3′-thio-deoxyinosine on a microarray. The oligonucleotide probes were synthesized from 3′-S-(2-cyanoethyl-N,N-diisopropylphosphorothioamidite)-5′-O-(4,4′ dimet-hoxytrityl) deoxyinosine. The resultant probes could be cleaved chemically by aqueous silver ions under mild conditions, generating a 5′-terminal phosphate group in a degradation oligodeoxynucleotide fragment. This 5′-terminal phosphate was used directly for detecting the corresponding bases in subsequent sequencing cycles. The queried bases in the template were identified with eight cycles of ligation and cleavage. The read length of our method could reach up to 40 bp with high accuracy. As this SBL method uses commercially available enzymes and standard microarrays, it will be amenable to automation and adaptation by the research community.
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Document Type: Research Article
Publication date: May 1, 2014
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