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Sequencing-by-Ligation Using Oligonucleotide Probes with 3′-Thio-Deoxyinosine

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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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Keywords: 3′-S-(2-CYANOETHYL-N,N-DIISOPROPYLPHOSPHOROTHIOAMIDITE)-5′-O-(4,4′ DIMETHOXYTRITYL)DEOXYINOSINE; 3′-THIO-DEOXYINOSINE; OLIGONUCLEOTIDE PROBE; SEQUENCING-BY-LIGATION (SBL)

Document Type: Research Article

Publication date: May 1, 2014

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  • Journal of Biomedical Nanotechnology (JBN) is a peer-reviewed multidisciplinary journal providing broad coverage in all research areas focused on the applications of nanotechnology in medicine, drug delivery systems, infectious disease, biomedical sciences, biotechnology, and all other related fields of life sciences.
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