DNA sequencing by denaturation: principle and thermodynamic simulations.
DNA sequencing by denaturation: principle and thermodynamic simulations.
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DOI：
10.1016/j.ab.2008.09.048
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发表时间：
2009-01-01
影响因子：
2.9
通讯作者：
Huang, Xiaohua
中科院分区：
文献类型：
--
作者：
Chen, Ying-Ja;Huang, Xiaohua
关键词：
我们描述了一种新的 DNA 测序方法，称为变性测序 (SBD)。在固体表面上的模板上进行桑格双脱氧测序反应，生成由荧光标记的双脱氧核糖核苷酸随机终止的 DNA 片段梯。标记的 DNA 片段依次从模板上变性，并通过测量表面荧光强度的变化来监控该过程。通过分析变性谱，可以确定模板的碱基序列。利用热力学原理，我们模拟了一系列 12 至 32 个碱基的寡核苷酸的变性曲线，并开发了一种碱基识别算法来解码序列。这些模拟表明，SBD 可以对多达 20 个碱基的 DNA 分子进行测序。溶液中 DNA 片段熔解曲线的实验测量证实 DNA 序列可以通过 SBD 确定。讨论了 SBD 的潜在局限性和优点。利用 SBD，可以使用极少量的测序试剂在表面的小区域上并行进行数百万次测序反应。因此，SBD DNA 测序可能会显着提高大规模基因组重测序的速度并降低成本。
We describe a new DNA sequencing method called sequencing by denaturation (SBD). A Sanger dideoxy sequencing reaction is performed on the templates on a solid surface to generate a ladder of DNA fragments randomly terminated by fluorescently-labeled dideoxyribonucleotides. The labeled DNA fragments are sequentially denatured from the templates and the process is monitored by measuring the change in fluorescence intensities from the surface. By analyzing the denaturation profiles, the base sequence of the template can be determined. Using thermodynamic principles, we simulated the denaturation profiles of a series of oligonucleotides ranging from 12 to 32 bases and developed a base-calling algorithm to decode the sequences. These simulations demonstrate that DNA molecules up to 20 bases can be sequenced by SBD. Experimental measurements of the melting profiles of DNA fragments in solution confirm that DNA sequence can be determined by SBD. The potential limitations and advantages of SBD are discussed. With SBD, millions of sequencing reactions can be performed on a small area on a surface in parallel with a very small amount of sequencing reagents. Therefore, DNA sequencing by SBD could potentially result in a significant increase in speed and reduction in cost in large-scale genome re-sequencing.
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