Fluorescent in situ sequencing on polymerase colonies
Fluorescent in situ sequencing on polymerase colonies
复制标题
DOI：
10.1016/s0003-2697(03)00291-4
复制
发表时间：
2003-09-01
影响因子：
2.9
通讯作者：
Church, GM
中科院分区：
文献类型：
--
作者：
Mitra, RD;Shendure, J;Church, GM
DNA分离、扩增和测序的整合可以通过使用聚合酶菌落（polonies）和荧光dNTP整合循环来实现。在本文中，我们提出了四项进展，使我们更接近成本有效地使用polony技术进行基因组测序。首先，聚合酶捕获技术使DNA聚合酶在聚丙烯酰胺基质中有效地扩展核苷酸，并消除了测序周期中酶的损失。接下来，我们提出了两种新型的可逆性染料标记的核苷酸类似物，表明DNA聚合酶可以结合这些类似物，并证明染料可以通过硫醇还原或光照去除。利用这些核苷酸，我们平行地对多个多倍体进行了测序。此外，我们发现，通过限制极性扩增反应中游离引物的浓度，可以在相邻极性之间最小重叠的情况下获得高密度的极性。最后，我们开发了自动图像对齐和序列调用软件。(C) 2003 Elsevier Science（美国）版权所有。
Integration of DNA isolation, amplification, and sequencing can be achieved by the use of polymerase colonies (polonies) and cycles of fluorescent dNTP incorporation. In this paper, we present four advances that bring us closer to sequencing genomes cost-effectively using the polony technology. First, a polymerase trapping technique enables efficient nucleotide extension by DNA polymerase in a polyacrylamide matrix and eliminates loss of enzyme during sequencing cycles. Next, we present two novel types of reversibly dye-labeled nucleotide analogues, show that DNA polymerase can incorporate these analogues, and demonstrate that the dyes can be removed by thiol reduction or light exposure. Using these nucleotides, we have sequenced multiple polonies in parallel. In addition, we have found that a high density of polonies can be achieved with minimal overlap between adjacent polonies by limiting the concentration of free primer in the polony amplification reactions. Finally, we have developed software for automated image alignment and sequence calling. (C) 2003 Elsevier Science (USA). All rights reserved.