PROGRESSIVE LIGATION-MEDIATED PCR SEQUENCING

我们建议进行一项可行性研究，以开发一种新的方法 我们称之为渐进连接介导的聚合酶的定向测序 链式反应(LM-PCR)测序。我们的方法应该提供一种方法 在基因组上向上或向下移动，从任何选定的点开始，而在 同时为以下两个主要瓶颈提供替代方案 大规模测序项目：即亚克隆和寡核苷酸 综合。使用我们提出的渐进式LM-PCR测序方法，(1) 应避免亚克隆，因为目标序列可能会被 连接介导的聚合酶链式反应，它允许人们扩增DNA的特定区域 仅由一种基因特异的聚合酶链式反应(Mueller和Wold，1989)确定； (2)应避免寡核苷酸合成，因为需要的是 可以通过以下方法从聚合酶链式反应扩增的DNA中分离出基因组 用IIS类限制性内切酶(它切割特定的DNA 距离识别地点的距离)。渐进式LM-PCR的概念 在以下情况下可能特别有用：定向测序方法 需要，例如，在猎枪方法中有效关闭 测序，从而便于组装和填补缺口的任务。 我们方法的独特之处在于，在放大 基因组的特定区域进行测序，我们将生成 测序该区域所需的模板和引物以及该引物 需要用来放大邻近区域。

We propose to conduct a feasibility study to develop a novel method for directed sequencing that we call Progressive Ligation-Mediated Polymerase Chain Reaction (LM-PCR) Sequencing. Our method should provide a way to walk up or down the genome, beginning at any selected point, while at the same time providing alternatives to two major bottlenecks associated with large scale sequencing projects: i.e., subcloning and oligonucleotide synthesis. With our proposed Progressive LM-PCR Sequencing method, (1) subcloning should be avoided because target sequences may be amplified by ligation-mediated PCR, which allows one to amplify specific regions of DNA defined by only one gene-specific PCR primer (Mueller and Wold, 1989); and (2) oligonucleotide synthesis should be avoided because the primers needed to walk down the genome may be isolated from the PCR-amplified DNA by digestion with class IIS restriction enzymes (which cleave DNA a specified distance from the recognition site). The concept of Progressive LM-PCR might be particularly useful where a method of directed sequencing is needed, e.g., for efficient closure in the shot-gun approach to sequencing, thereby facilitating the task of assembly and gap-filling. The unique feature of our method is that, in the process of amplifying a specific region of a genome for sequencing, we would generate the templates and primers needed to sequence the region as well as the primers needed to amplify the adjoining region.