SHOTGUN SEQUENCING OF MULTIPLE FULL LENGTH CDNA CLONES

本研究的目的是开发和简化有效的方法， 全长cDNA插入片段的快速准确测序， 随着大量新基因的出现， 在人类基因组计划中被发现。 一种同时 鸟枪测序的多个cDNA克隆选择高质量的 “Soares”婴儿脑cDNA文库（INIB）将用于产生 100-300个独特的人cDNA克隆的完整序列。 每一个克隆人都将 微生物制备，插入物将通过用 限制性内切酶，然后凝胶纯化。 猎枪图书馆将 使用改进的“双适配器”方法构建。 cDNA插入物 将以20-100个的池合并，并通过连接或形式连接 大约30-150 kb的DNA片段。 多联体将被剪切， 随机克隆到M13载体上。 随机测序后， 序列将在组装阶段使用限制性内切酶进行鉴定 位点作为每个克隆末端的“标签”，而剩余的缺口和 完全双链覆盖将通过使用引物- 定向测序 本研究将提供有用的人cDNA序列 这将导致简化方法， 快速产生全长cDNA克隆序列。

The aim of this study is to develop and streamline efficient approaches for the rapid and accurate sequencing of full-length cDNA inserts, which is becoming increasingly important with the large number of novel genes discovered within the Human Genome Project. A strategy for simultaneous shotgun sequencing of multiple cDNA clones selected from high quality "Soares" infant brain cDNA library (INIB) will be used to generate the complete sequences of 100-300 unique human cDNA clones. Each clone will be prepared microbially and the inserts will be isolated by digestion with restriction enzymes followed by gel-purification. The shotgun library will be constructed using an improved "double adaptor" method. cDNA inserts will be combined in pools of 20-100 and concatenated by ligation ot form approximately 30-150 kb DNA fragments. The concatamers will be sheared and randomly cloned to M13 vector. After random sequencing, individual cDNA sequence will be identified at the assembly stage using restriction enzyme sites as "tags" for the ends of each clone, while remaining gaps and complete double-stranded coverage will be completed by using primer- directed sequencing. This study will provide useful human cDNA sequence information and it will result in streamlining of the methodology to rapidly generate full-length cDNA clone sequences.