DC Field | Value | Language |
---|---|---|
dc.contributor.author | SEO, TAE SEOK | ko |
dc.contributor.author | Bai, XP | ko |
dc.contributor.author | Ruparel, H | ko |
dc.contributor.author | Li, ZM | ko |
dc.contributor.author | Turro, NJ | ko |
dc.contributor.author | Ju, JY | ko |
dc.date.accessioned | 2009-05-06T02:54:11Z | - |
dc.date.available | 2009-05-06T02:54:11Z | - |
dc.date.created | 2012-02-06 | - |
dc.date.created | 2012-02-06 | - |
dc.date.issued | 2004-04 | - |
dc.identifier.citation | PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.101, no.15, pp.5488 - 5493 | - |
dc.identifier.issn | 0027-8424 | - |
dc.identifier.uri | http://hdl.handle.net/10203/8851 | - |
dc.description.abstract | DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence. | - |
dc.description.sponsorship | National Science Foundation Sensing and Imaging Initiative Grant 0097793, a Packard Fellowship for Science and Engineering, Center of Excellence in Genomic Science Grant P50 HG002806 from National Institutes of Health | en |
dc.language | English | - |
dc.language.iso | en_US | en |
dc.publisher | NATL ACAD SCIENCES | - |
dc.subject | COPPER(I)-CATALYZED AZIDE-ALKYNE | - |
dc.subject | MASS-SPECTROMETRY | - |
dc.subject | TERMINAL ALKYNES | - |
dc.subject | HUMAN-GENOME | - |
dc.subject | CYCLOADDITION | - |
dc.subject | GENE | - |
dc.title | Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry | - |
dc.type | Article | - |
dc.identifier.wosid | 000220861500036 | - |
dc.identifier.scopusid | 2-s2.0-1842631432 | - |
dc.type.rims | ART | - |
dc.citation.volume | 101 | - |
dc.citation.issue | 15 | - |
dc.citation.beginningpage | 5488 | - |
dc.citation.endingpage | 5493 | - |
dc.citation.publicationname | PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | - |
dc.identifier.doi | 10.1073/pnas.0401138101 | - |
dc.embargo.liftdate | 9999-12-31 | - |
dc.embargo.terms | 9999-12-31 | - |
dc.contributor.localauthor | SEO, TAE SEOK | - |
dc.contributor.nonIdAuthor | Bai, XP | - |
dc.contributor.nonIdAuthor | Ruparel, H | - |
dc.contributor.nonIdAuthor | Li, ZM | - |
dc.contributor.nonIdAuthor | Turro, NJ | - |
dc.contributor.nonIdAuthor | Ju, JY | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordPlus | COPPER(I)-CATALYZED AZIDE-ALKYNE | - |
dc.subject.keywordPlus | MASS-SPECTROMETRY | - |
dc.subject.keywordPlus | TERMINAL ALKYNES | - |
dc.subject.keywordPlus | HUMAN-GENOME | - |
dc.subject.keywordPlus | CYCLOADDITION | - |
dc.subject.keywordPlus | GENE | - |
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