The goals of this proposal, in response to the PA for mapping, DNA sequencing, and technology development in support of the human genome program, are twofold: (i) to develop a novel method that will facilitate the sequencing of unknown DNA flanking a known site in human genomic DNA; (ii) to apply this method to the cloning of the ends of inserts (endpoints) of human genome fragments that have been subcloned into yeast artificial chromosome (YAC) vectors. Cloning of YAC endpoints will permit the generation of an overlapping set of YAC clones, so that sequence information obtained from individual YAC clones can be aligned. These goals will be accomplished by a novel method comprising a primer dependent attachment of a known sequence to the uncharacterized side of that specific DNA strand which contains the unknown sequence. This will permit specific PCR amplification of the unknown DNA because a known sequence now flanks the strand that contains the unknown DNA. The PCR template is generated by the following steps: 1) Digestion of genomic DNA with a restriction enzyme that leaves a 5' overhang. 2) Ligation of a single-stranded oligonucleotide to the restriction enzyme digested genomic DNA. This oligonucleotide is designed to be complementary to the known region of DNA immediately upstream from the unknown region of DNA. 3) Denaturation and self-annealing under dilute conditions, resulting in strands of genomic DNA which contain the complement to the ligated piece to form a stem-loop structure. The sequence specific annealing that constitutes the stem can prime template-directed DNA polymerization from the ligated oligonucleotide. This polymerization results in known DNA being placed on the uncharacterized end of the unknown DNA contained in the loop. Following the generation of this template, application of PCR using known sequence that now flanks the unknown DNA permits specific amplification of the unknown DNA. Preliminary data provide evidence that this approach will be effective. Implementation of this method will permit the amplification and sequencing of unknown flanking DNA directly from the human genome. Furthermore, in the context of cloning technology, it will facilitate the generation of contiguous YAC clones by providing a rapid and reliable method for the cloning of YAC endpoints. Therefore, this method will have broad applicability in the human genome project. It will permit the ordering of large fragments of individual chromosomes contained in YAC vectors, so that sequences obtained from distant regions of a chromosome can be aligned. This method also has the more ambitious potential of bypassing cloning altogether in obtaining sequence information from the human genome. This will lessen the number of steps in obtaining sequencing template, bypass cloning artifacts, and permit the sequencing of unclonable DNA.
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