Probe optimization for sequencing by ligation.
Identifieur interne : 001614 ( Main/Exploration ); précédent : 001613; suivant : 001615Probe optimization for sequencing by ligation.
Auteurs : Dan Pu [République populaire de Chine] ; Jing Chen [République populaire de Chine] ; Xiaoting Qian [République populaire de Chine] ; Pengfeng Xiao [République populaire de Chine]Source :
- Journal of biochemistry [ 1756-2651 ] ; 2015.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical : Fluorescent Dyes.
- methods : Oligonucleotide Array Sequence Analysis.
Abstract
Sequencing by ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled fluorescent probes. Second, probes between 7- and 9-mers in length were designed to uniform Tm difference. We hoped the uniformed probes with smaller Tm difference could improve the ligation efficiency. However, 8-mer probes with larger Tm difference showed stronger fluorescence intensities. Third, we evaluated whether probes containing deoxyinosines either in the 5' or the 3' end had influence on the ligation efficiency. Consequently, probes containing deoxyinosines at the 5' termini might decrease the ligation efficiency, and the accumulation of 3' terminal deoxyinosines in the sequencing primers was likely to reduce the fluorescence intensity and the ligation efficiency, which was inconsistent with the traditional viewpoint. The optimized probes will improve the ligation efficiency and accuracy in SBL.
DOI: 10.1093/jb/mvu082
PubMed: 25538255
Affiliations:
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210096, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096</wicri:regionArea><wicri:noRegion>Nanjing 210096</wicri:noRegion></affiliation></author><author><name sortKey="Qian, Xiaoting" sort="Qian, Xiaoting" uniqKey="Qian X" first="Xiaoting" last="Qian">Xiaoting Qian</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096</wicri:regionArea><wicri:noRegion>Nanjing 210096</wicri:noRegion></affiliation></author><author><name sortKey="Xiao, Pengfeng" 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China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096</wicri:regionArea><wicri:noRegion>Nanjing 210096</wicri:noRegion></affiliation></author><author><name sortKey="Chen, Jing" sort="Chen, Jing" uniqKey="Chen J" first="Jing" last="Chen">Jing Chen</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096</wicri:regionArea><wicri:noRegion>Nanjing 210096</wicri:noRegion></affiliation></author><author><name sortKey="Qian, Xiaoting" sort="Qian, Xiaoting" uniqKey="Qian X" first="Xiaoting" last="Qian">Xiaoting Qian</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096</wicri:regionArea><wicri:noRegion>Nanjing 210096</wicri:noRegion></affiliation></author><author><name sortKey="Xiao, Pengfeng" sort="Xiao, Pengfeng" uniqKey="Xiao P" first="Pengfeng" last="Xiao">Pengfeng Xiao</name><affiliation wicri:level="1"><nlm:affiliation>State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China xiaopf@seu.edu.cn.</nlm:affiliation><country wicri:rule="url">République populaire de 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scheme="MESH" xml:lang="fr"><term>Colorants fluorescents</term><term>Séquençage par oligonucléotides en batterie</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Sequencing by ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled fluorescent probes. Second, probes between 7- and 9-mers in length were designed to uniform Tm difference. We hoped the uniformed probes with smaller Tm difference could improve the ligation efficiency. However, 8-mer probes with larger Tm difference showed stronger fluorescence intensities. Third, we evaluated whether probes containing deoxyinosines either in the 5' or the 3' end had influence on the ligation efficiency. Consequently, probes containing deoxyinosines at the 5' termini might decrease the ligation efficiency, and the accumulation of 3' terminal deoxyinosines in the sequencing primers was likely to reduce the fluorescence intensity and the ligation efficiency, which was inconsistent with the traditional viewpoint. The optimized probes will improve the ligation efficiency and accuracy in SBL. </div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><country name="République populaire de Chine"><noRegion><name sortKey="Pu, Dan" sort="Pu, Dan" uniqKey="Pu D" first="Dan" last="Pu">Dan Pu</name></noRegion><name sortKey="Chen, Jing" sort="Chen, Jing" uniqKey="Chen J" first="Jing" last="Chen">Jing Chen</name><name sortKey="Qian, Xiaoting" sort="Qian, Xiaoting" uniqKey="Qian X" first="Xiaoting" last="Qian">Xiaoting Qian</name><name sortKey="Xiao, Pengfeng" sort="Xiao, Pengfeng" uniqKey="Xiao P" first="Pengfeng" last="Xiao">Pengfeng Xiao</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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