Building upon the complete sequence of the human genome, intense efforts are currently underway to identify the underlying genetic link to common diseases by single nucleotide polymorphism (SNP) mapping or direct association. Technology development focused on rapid, high-throughput, and low cost SNP detection would represent a major advance for the application of genetic information in applied medicine.
The aim of the R41 proposal is the development of the Base Addition Sequencing Scheme (BASS) with potential application for targeted mini-sequencing from PCR-amplified from genomic DNA materials. We have demonstrated the feasibility of BASS and have identified the major challenges in development for DNA sequencing applications. Namely, they are (a) non-uniform UV deprotection of the different 2-nitrobenzyl nucleobase triphosphates and (b) poor incorporation efficiencies of 3'-(substituted-2-nitrobenzyl)-dNTPs by commercially available DNA polymerases. To overcome these obstacles, we propose the synthesis of '3-0- (substituted-2-nitrobenzyl)-dNTPs, which show uniform deprotection efficiencies for the four nucleobases and the identification of novel Taq DNA polymerase variants by random mutagenesis screens. The test of feasibility for the BASS technology will be two cycles of stepwise DNA synthesis (that is, two rounds of incorporation and deprotection) in all sixteen 2-base extension combinations. The outcome of this research will provide a more accurate estimate of the cycle efficiency, which will be an indicator of the sequence readlength. Successful implementation of these aims will result would demonstrate the BASS technology for application of de novo SNP discovery.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Small Business Technology Transfer (STTR) Grants - Phase I (R41)
Project #
5R41HG003072-02
Application #
6805134
Study Section
Special Emphasis Panel (ZRG1-SSS-Y (10))
Program Officer
Schloss, Jeffery
Project Start
2003-09-30
Project End
2004-09-29
Budget Start
2004-08-01
Budget End
2004-09-29
Support Year
2
Fiscal Year
2004
Total Cost
$54,488
Indirect Cost
Name
Lasergen
Department
Type
DUNS #
106700375
City
Houston
State
TX
Country
United States
Zip Code
77054
Metzker, Michael L (2010) Sequencing technologies - the next generation. Nat Rev Genet 11:31-46
Wu, Weidong; Stupi, Brian P; Litosh, Vladislav A et al. (2007) Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates. Nucleic Acids Res 35:6339-49
Metzker, Michael L (2005) Emerging technologies in DNA sequencing. Genome Res 15:1767-76