DNA SEQUENCING--ITERATIVE STEPS BY PRODUCT REGENERATION

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DESCRIPTION (Adapted from the Investigator's Abstract): In this grant application, the investigator proposes to develop a method that can sequence many DNA fragments in parallel without gel resolution of DNA fragments. DNA sequencing currently uses the Sanger method, requiring a gel resolution step that limits output and accuracy. Sequencing by hybridization is hindered by inverted repeats in the template and sequence composition effects on duplex formation. The method presented here sequences DNA that is almost entirely double-stranded, preventing sequence errors resulting from DNA secondary structures. The first specific aim is to develop a protocol that can sequence 150 nucleotides in each of many vector inserts in parallel. This method uses ligation of an adaptor that encodes the recognition domain for a class-IIS restriction endonuclease and contains a sequence unique to each sequencing cycle. PCR seamlessly appends the restriction endonuclease recognition domain, and digestion with a class-IIS restriction endonuclease that recognizes the appended recognition domain trims the DNA segment, generating a DNA template composed of a short overhang. Sequencing of a nucleotide in the overhang template occurs through template-directed polymerization with labeled ddNTPs or by template-directed ligation to labeled adaptors. The ligated adaptor sequence that is unique to each cycle allows the PCR step to exponentially regenerate only the desired template precursor during each iteration; thus, this grant tests the hypothesis that exponential DNA amplification in vitro can overcome the background signal accumulation that disables alternative iterative DNA sequencing methods. Also, DNA sequencing occurs in strides up to the number of nucleotides separating the recognition and cleavage domain, maximizing the span of DNA sequenced for a given number of nucleotides. The second specific aim is to develop a protocol to block pre-existing internal class-IIS recognition domains in the DNA segment being sequenced. Using existing instruments and microtiter plates, this method will be able to sequence greater than 1000 vector inserts concurrently, reading greater than 100,000 nucleotides. In the future this method can be adapted to a biochip format. The work proposed in this grant application will allow the sequencing of at least 150 nucleotides in each of thousands of inserts in a DNA library. The sequenced segments will be correct and of sufficient length for extensive bottom up genome sequence reconstruction.