PROGRESSIVE LIGATION-MEDIATED PCR SEQUENCING

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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.