Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.
Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.
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细菌 DNA 的直接基因组测序：大肠杆菌的丙酮酸激酶 I 基因。
DOI：
10.1073/pnas.86.18.6883
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发表时间：
1989
影响因子：
11.1
通讯作者：
Gilbert,W
中科院分区：
文献类型：
--
作者：
Ohara,O;Dorit,RL;Gilbert,W
基因组测序程序通过“多重步移”方法应用于细菌 DNA 未表征区域的直接测序。用各种限制性酶切割大量大肠杆菌 DNA 样品，进行化学测序降解，在测序凝胶中运行，然后转移到尼龙膜上。当标记的寡聚体与膜杂交时，只要探针位于限制性酶切附近，就会出现序列梯。然后根据超出其他限制性位点的序列合成新探针，并对膜进行重新探测以揭示新序列。寡聚物探针合成和随后的重新探测的重复循环允许沿着基因组快速序列行走。这种寡聚物步移技术用于对大肠杆菌中的丙酮酸激酶 (EC 2.7.1.40) 基因进行测序，而无需采用克隆或文库构建。通过聚合酶链式反应扩增测序区域，随后使用体内和体外系统进行转录和翻译，所得基因产物的特征在于显示该基因编码丙酮酸激酶的I型同工型。
The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.