Abstract

The objective of the proposed research is to develop an integrated system for DNA sequencing by synthesis (SBS) using cleavable fluorescent nucleotide reversible terminators. We will (1) design and synthesize a library of 2'-deoxynucleotide analogues that each consists of a unique fluorescent dye attached to the base through a cleavable linker, and whose 3'-OH group is modulated as a reversible terminator in polymerase reaction. These nucleotide analogues are designed in such a way that after they are incorporated into the growing strand of DNA in the polymerase reaction, the fluorescent dyes are cleaved by irradiation at 355 nm or by specific chemical reagents in a mild condition that is compatible with DNA, and the 3'-OH can be regenerated to continue the polymerase reaction; (2) develop an """"""""ePCR-bead-on-chip (ePBC)"""""""" approach for amplifying and immobilizing DNA products for sequencing by synthesis (SBS). The ePBC approach is based on emulsion PCR on beads and then covalently immobilizing the beads, each containing many copies of unique amplified DNA templates on a chip; (3) evaluate and implement a 4-color SBS system consisting of a laser induced photolysis device, a liquid handling module, and a high sensitivity CCD based 4-color fluorescence imager for sequencing a variety of DNA templates; (4) develop a library construction method for pair-end sequencing of DNA clones using the sequencing chemistry developed in aim 1, the ePBC DNA chip that is developed in aim 2, and the 4-color SBS system in aim 3 for sequencing BAC clones from bacteria and other model organisms. This new system for genome sequencing and resequencing will have wide applications in biology and biomedical research. ? ? ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG003582-06
Application #
7477305
Study Section
Special Emphasis Panel (ZHG1-HGR-N (M1))
Program Officer
Schloss, Jeffery
Project Start
2004-09-29
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2010-07-31
Support Year
6
Fiscal Year
2008
Total Cost
$1,087,635
Indirect Cost

  Institution

Name
Columbia University (N.Y.)
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027

  Publications

Guo, Jia; Yu, Lin; Turro, Nicholas J et al. (2010) An integrated system for DNA sequencing by synthesis using novel nucleotide analogues. Acc Chem Res 43:551-63
Guo, Jia; Xu, Ning; Li, Zengmin et al. (2008) Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc Natl Acad Sci U S A 105:9145-50
Chen, Lu; Rengifo, Hernan R; Grigoras, Cristian et al. (2008) Spin-on end-functional diblock copolymers for quantitative DNA immobilization. Biomacromolecules 9:2345-52
Wu, Jian; Zhang, Shenglong; Meng, Qinglin et al. (2007) 3'-O-modified nucleotides as reversible terminators for pyrosequencing. Proc Natl Acad Sci U S A 104:16462-7
Meng, Qinglin; Kim, Dae Hyun; Bai, Xiaopeng et al. (2006) Design and synthesis of a photocleavable fluorescent nucleotide 3'-O-allyl-dGTP-PC-Bodipy-FL-510 as a reversible terminator for DNA sequencing by synthesis. J Org Chem 71:3248-52
Bi, Lanrong; Kim, Dae Hyun; Ju, Jingyue (2006) Design and synthesis of a chemically cleavable fluorescent nucleotide, 3'-O-allyl-dGTP-allyl-bodipy-FL-510, as a reversible terminator for DNA sequencing by synthesis. J Am Chem Soc 128:2542-3
Ju, Jingyue; Kim, Dae Hyun; Bi, Lanrong et al. (2006) Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proc Natl Acad Sci U S A 103:19635-40
Bai, Xiaopeng; Edwards, John; Ju, Jingyue (2005) Molecular engineering approaches for DNA sequencing and analysis. Expert Rev Mol Diagn 5:797-808
Seo, Tae Seok; Bai, Xiaopeng; Kim, Dae Hyun et al. (2005) Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. Proc Natl Acad Sci U S A 102:5926-31
Ruparel, Hameer; Bi, Lanrong; Li, Zengmin et al. (2005) Design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis. Proc Natl Acad Sci U S A 102:5932-7