title: Probe optimization for sequencing by ligation.
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.
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Pu
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Qian
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China xiaopf@seu.edu.cn.
authors.forename: Pengfeng
authors.initials: P
authors.lastname: Xiao
mesh_terms: D005456:Fluorescent Dyes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes
keywords: deoxyinosine; double-labelled fluorescent probe; high-throughput sequencing; optimization; probe optimization; sequencing by ligation (SBL)