A RAPID SINGLE-STRANDED CLONING STRATEGY FOR PRODUCING A SEQUENTIAL SERIES OF OVERLAPPING CLONES FOR USE IN DNA SEQUENCING - APPLICATION TO SEQUENCING THE CORN MITOCHONDRIAL 18-S RDNA
A RAPID SINGLE-STRANDED CLONING STRATEGY FOR PRODUCING A SEQUENTIAL SERIES OF OVERLAPPING CLONES FOR USE IN DNA SEQUENCING - APPLICATION TO SEQUENCING THE CORN MITOCHONDRIAL 18-S RDNA
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DOI：
10.1016/0147-619x(85)90053-8
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发表时间：
1985-01-01
期刊：
影响因子：
2.6
通讯作者：
HOUCHINS, JP
中科院分区：
文献类型：
--
作者：
DALE, RMK;MCCLURE, BA;HOUCHINS, JP
描述了一种简单的新程序，用于产生用于 DNA 测序的一系列连续的重叠克隆。该技术使用单链 M13 DNA 和互补的 [c] DNA 寡聚体来形成特异性切割和连接底物。它与克隆到载体中的 DNA 序列无关。通过 T4 DNA 聚合酶的 3'' 至 5'' 核酸外切酶活性，从插入片段的一端产生不同大小的缺失。缺失的大致大小和 DNA 测序的起始点可以通过亚克隆噬菌体 DNA 在琼脂糖凝胶上的电泳来估计。这大大减少了为获得完整序列而必须测序的模板数量。整个过程可以在不到 1 天内在 1 个试管中完成。该程序用于在 < 1 周内对玉米线粒体 18 S r[核糖体]DNA 和 5'' 侧翼区域（2622 个碱基）进行亚克隆和测序。讨论了寡聚物和单链 DNA 在插入、缺失和 cDNA 构建中的其他应用。
A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary [c] DNA oligomers to form specific cleavage and ligation substrates. It was independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3'' to 5'' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on an agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in 1 tube in < 1 day. The procedure was used to subclone and sequence the maize mitochondrial 18 S r[ribosomal]DNA and 5'' flanking region (2622 bases) in < 1 wk. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions and cDNAs are discussed.