DNA sequencing by hybridization using semi-degenerate bases.
DNA sequencing by hybridization using semi-degenerate bases.
复制标题
使用半简并碱基杂交进行 DNA 测序。
DOI：
10.1089/cmb.2004.11.753
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Oliver,JohnS
中科院分区：
文献类型：
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作者：
Preparata,FrancoP;Oliver,JohnS
增强用于DNAde novosequencing的杂交微阵列的性能的一种方法是使用具有未采样位置的间隙的探测图案。理想情况下，这样的缺口可以通过在微阵列寡核苷酸（探针）中包含称为通用碱基（与天然碱基非特异性结合）的通配符化合物来实现。建议的替代方案是在差距位置部署简并碱基，即，四种天然碱基的均匀混合物，随之而来的是杂交信号的恶化。在本文中，我们表明，这样的信号损失是一个小的缺点，与事实相比，退化基地不能被视为普遍的。实际上，杂交能量在任何微阵列特征处的实质性分布使得绝大多数错配比法律的匹配更强地结合。然而，我们观察到，在相同强度等级（A-T和C-G）的碱基对表现出更窄的能量范围。我们称半简并的间隙位置实现了与基地在相同的能源类，并表明，众所周知的序列重建算法可以修改，以实现测序效率的大幅改善。例如，使用49个特征的微阵列和可接受的杂交信号减弱，可以实现约4，000个碱基的长度（与标准均匀方法的< 250个碱基相比）。我们的方法还结合了使用的光谱表示观察到的特征熔化温度（模拟光谱），而不是直接在生化水平（数字光谱）的二元决策。虽然通用碱基代表了杂交测序的最终目标，但半天然碱基是已知最有效的替代物。
One way to enhance the performance of hybridization microarrrays for DNAde novosequencing is the use of probing patterns with gaps of unsampled positions. Ideally, such gaps could be realized by the inclusion into microarray oligos (probes) of wild-card compounds, referred to as universal bases (which bind nonspecifically to natural bases). The suggested alternative is to deploy in the gap positions degenerate bases, i.e., uniform mixtures of the four natural bases, with ensuing deterioration of the hybridization signal. In this paper, we show that such signal loss is a minor shortcoming, compared with the fact that degenerate bases cannot be treated as universal. Indeed, the substantial spread of hybridization energies at any microarray feature is such that on overwhelming number of mismatches bind more strongly than legal matches. We observed, however, that much narrower energy spreads are exhibited by pairs of bases in the same strength class (A-T and C-G). We callsemi-degeneratea gap position realized with bases in the same energy class and show that well-known sequence reconstruction algorithms can be modified to achieve substantial improvements in sequencing effectiveness. For example, with a 49-feature microarray and an acceptable weakening of the hybridization signal, one may achieve lengths of about 4,000 bases (compared with < 250 of the standard uniform method). Our approach also incorporates the use of a spectrum expressed in terms of observed feature melting temperatures (analog spectrum), rather than binary decisions made directly at the biochemical level (digital spectrum). While universal bases represent the ultimate goal of sequencing by hybridization, semidegenerate natural bases are the most effective known substitute.