Abstract

In view of the utilization of human genome data the capability of sequencing the genetic information of single cells without prior amplification would potentiate speed and sensitivity of gene function recognition. We propose to carry out single molecule sequencing by following the stepwise addition of base pairs during production of the complementary DNA strand of a given (unknown) template strand by the enzyme DNA polymerase. The incorporation of each base at the polymerase active site is to be recognized by detection of a specific fluorescent marker. Preliminary studies on DNA synthesis on immobilized template strands utilized the fluorescent dye PicoGreen (Molecular Probes Inc.), and it is shown that individual molecules of newly synthesized DNA can be detected. Next, we attempt to detect the separate binding events of PicoGreen molecules to the newly generated DNA with a fluorescence correlation spectroscopy setup. The DNA sequencing approach will also utilize nanofabricated compon ents for DNA manipulation and possible fluid flow.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR004224-14
Application #
6494051
Study Section
Project Start
2001-09-01
Project End
2002-08-31
Budget Start
Budget End
Support Year
14
Fiscal Year
2001
Total Cost
Indirect Cost

  Institution

Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850

  Publications

Migone, Fernando F; Cowan, Robert G; Williams, Rebecca M et al. (2016) In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation. Proc Natl Acad Sci U S A 113:2294-9
O'Dell, Ryan S; Cameron, David A; Zipfel, Warren R et al. (2015) Reelin Prevents Apical Neurite Retraction during Terminal Translocation and Dendrite Initiation. J Neurosci 35:10659-74
Byrnes, Laura J; Singh, Avtar; Szeto, Kylan et al. (2013) Structural basis for conformational switching and GTP loading of the large G protein atlastin. EMBO J 32:369-84
Jain, Manu; Robinson, Brian D; Scherr, Douglas S et al. (2012) Multiphoton microscopy in the evaluation of human bladder biopsies. Arch Pathol Lab Med 136:517-26
Degala, Satish; Williams, Rebecca; Zipfel, Warren et al. (2012) Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture. J Orthop Res 30:793-9
O'Dell, Ryan S; Ustine, Candida J M; Cameron, David A et al. (2012) Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone. Neural Dev 7:25
McMullen, J D; Kwan, A C; Williams, R M et al. (2011) Enhancing collection efficiency in large field of view multiphoton microscopy. J Microsc 241:119-24
Bowles, Robby D; Williams, Rebecca M; Zipfel, Warren R et al. (2010) Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disc composite via collagen gel contraction. Tissue Eng Part A 16:1339-48
McMullen, Jesse D; Zipfel, Warren R (2010) A multiphoton objective design with incorporated beam splitter for enhanced fluorescence collection. Opt Express 18:5390-8
Kim, Sally A; Sanabria, Hugo; Digman, Michelle A et al. (2010) Quantifying translational mobility in neurons: comparison between current optical techniques. J Neurosci 30:16409-16

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