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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG000569-02
Application #
3333751
Study Section
Genome Study Section (GNM)
Project Start
1992-03-15
Project End
1995-02-28
Budget Start
1993-03-01
Budget End
1994-02-28
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Francis, Owen E; Bendall, Matthew; Manimaran, Solaiappan et al. (2013) Pathoscope: species identification and strain attribution with unassembled sequencing data. Genome Res 23:1721-9
Cohen, Adam L; Piccolo, Stephen R; Cheng, Luis et al. (2013) Genomic pathway analysis reveals that EZH2 and HDAC4 represent mutually exclusive epigenetic pathways across human cancers. BMC Med Genomics 6:35
Piccolo, Stephen R; Sun, Ying; Campbell, Joshua D et al. (2012) A single-sample microarray normalization method to facilitate personalized-medicine workflows. Genomics 100:337-44
Jones, Douglas H; Winistorfer, Stanley C (2003) Recombination and site-directed mutagenesis using recombination PCR. Methods Mol Biol 226:517-24
Jones, D H; Winistorfer, S C (1997) Amplification of 4-9-kb human genomic DNA flanking a known site using a panhandle PCR variant. Biotechniques 23:132-8
Jones, D H; Winistorfer, S C (1997) Recombination and site-directed mutagenesis using recombination PCR. Methods Mol Biol 67:131-40
Jones, D H (1995) Panhandle PCR. PCR Methods Appl 4:S195-201
Jones, D H; Riley, A N; Winistorfer, S C (1994) Production of a vector to facilitate DNA mutagenesis and recombination. Biotechniques 16:694-701
Jones, D H (1994) PCR mutagenesis and recombination in vivo. PCR Methods Appl 3:S141-8
Jones, D H; Winistorfer, S C (1993) A method for the amplification of unknown flanking DNA: targeted inverted repeat amplification. Biotechniques 15:894-904

Showing the most recent 10 out of 11 publications