An approach to acquiring DNA sequence data is proposed that involves the imaging of single DNA molecules. Specific or random priming sites throughout the molecules will be tested for their ability to have a fluorescent nucleotide added by a polymerase. An imaging system will detect the single fluorochromes and document the next base in the sequence series for each site. Potential acquisition of 20 Kb of DNA sequence data contained within 10-40 digital images is described. The proposed potential rate of sequence acquisition is 180 Kb per hr. A similar approach to detecting single base polymorphisms is proposed that does not require directly detecting a single molecule, but instead tests whether multiple labeled nucleotides are added to a site that may or may not terminate with a dideoxynucleotide. A plan to map and subclone BACs as a paradigm for eventual DNA sequencing is proposed. A plan to improve charge density on glass surfaces as a way to optimize DNA fixation for optical mapping is also proposed.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
7R01HG000225-09
Application #
2889643
Study Section
Special Emphasis Panel (ZRG2-BIOL-1 (01))
Program Officer
Schloss, Jeffery
Project Start
1991-01-01
Project End
2004-03-31
Budget Start
1999-09-30
Budget End
2004-03-31
Support Year
9
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Krerowicz, Samuel J W; Hernandez-Ortiz, Juan P; Schwartz, David C (2018) Microscale Objects via Restructuring of Large, Double-Stranded DNA Molecules. ACS Appl Mater Interfaces :
Kounovsky-Shafer, Kristy L; Hernandez-Ortiz, Juan P; Potamousis, Konstantinos et al. (2017) Electrostatic confinement and manipulation of DNA molecules for genome analysis. Proc Natl Acad Sci U S A 114:13400-13405
Lequieu, Joshua; Schwartz, David C; de Pablo, Juan J (2017) In silico evidence for sequence-dependent nucleosome sliding. Proc Natl Acad Sci U S A 114:E9197-E9205
Lequieu, Joshua; Córdoba, Andrés; Schwartz, David C et al. (2016) Tension-Dependent Free Energies of Nucleosome Unwrapping. ACS Cent Sci 2:660-666
Li, Yang; Zhou, Shiguo; Schwartz, David C et al. (2016) Allele-Specific Quantification of Structural Variations in Cancer Genomes. Cell Syst 3:21-34
Mendelowitz, Lee M; Schwartz, David C; Pop, Mihai (2016) Maligner: a fast ordered restriction map aligner. Bioinformatics 32:1016-22
Park, Dong-Wook; Kim, Hyungsoo; Bong, Jihye et al. (2016) Flexible bottom-gate graphene transistors on Parylene C substrate and the effect of current annealing. Appl Phys Lett 109:152105
Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon et al. (2016) DNA binding fluorescent proteins for the direct visualization of large DNA molecules. Nucleic Acids Res 44:e6
Zhou, Shiguo; Goldstein, Steve; Place, Michael et al. (2015) A clone-free, single molecule map of the domestic cow (Bos taurus) genome. BMC Genomics 16:644
Hernández-Ortiz, Juan P; de Pablo, Juan J (2015) Self-consistent description of electrokinetic phenomena in particle-based simulations. J Chem Phys 143:014108

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