USA flag logo/image

An Official Website of the United States Government

Ex Cyto DNA Sequencing from Single Bacterial Colonies

Award Information

Agency:
Department of Health and Human Services
Branch:
N/A
Award ID:
80238
Program Year/Program:
2009 / SBIR
Agency Tracking Number:
HG004095
Solicitation Year:
N/A
Solicitation Topic Code:
N/A
Solicitation Number:
N/A
Small Business Information
LUCIGEN CORPORATION
2905 PARMENTER ST. Middleton, WI 53562-2547
View profile »
Woman-Owned: No
Minority-Owned: No
HUBZone-Owned: No
 
Phase 2
Fiscal Year: 2009
Title: Ex Cyto DNA Sequencing from Single Bacterial Colonies
Agency: HHS
Contract: 2R44HG004095-02
Award Amount: $750,000.00
 

Abstract:

DESCRIPTION (provided by applicant): The long term objective of this research is to develop improved DNA polymerases that will substantially advance Sanger and next generation sequencing technologies. The primary goal of the Phase I research was to deve lop a rapid and inexpensive method to perform Sanger sequencing directly from single bacterial colonies. This method bypasses the time and expense of template purification needed for conventional DNA sequencing. It is based on the development of a modified Taq fusion DNA polymerase (DNAP) with improved DNA affinity and processivity. Chromosomal integration and expression of such a polymerase into host cells used for cloning should enable a new concept, termed Ex cyto sequencing . Analogous to colony PCR, E x cyto sequencing will eliminate the overnight growth of bacterial cultures, expensive template purification, and the purchase of purified DNA polymerase. In the Phase I research we developed a new fusion polymerase with novel enzymatic attributes, which w as shown to improve multiple aspects of nucleic acid sequencing and amplification. In addition to sequencing trace amounts of DNA from a single bacterial colony, the new Taq fusion polymerase enabled the following procedures: sequencing difficult templates unresolved by other enzymes, sequencing directly from liquid cultures using as little as 5 ul of outgrowth media, tight DNA binding in a variety of buffer conditions, and long PCR (10 kb). The specific aims of the Phase II proposal are to complete the dev elopment and optimization of Ex cyto sequencing and to modify other DNA polymerases used in next generation sequencing, such as Bst, Klenow, and T4 DNAPs. These improved enzymes should provide superior read lengths and the ability to sequence through diffi cult structures, thereby improving the accuracy of base calling and the subsequent assembly of genomes. These enzymes will improve numerous nucleic acid synthesis applications, such as emulsion PCR, used for the in vitro clonal amplification of templates, whole genome amplification, long PCR amplification, and a host of other methods that are constrained by existing DNAP capabilities. PUBLIC HEALTH RELEVANCE: The success of the human genome project has spawned explosive growth in the demand for DNA sequence information. The discovery of new genes from a variety of species will have a large impact on understanding human health and disease. Despite improvements in speed and reduction in costs of DNA sequence analysis, the process is still time, labor, and cost-intensive. This proposal seeks to dramatically improve the speed while decreasing the costs of DNA sequencing by developing a next generation DNA polymerase.

Principal Investigator:

David A. Mead
6088319011
DMEAD@LUCIGEN.COM

Business Contact:


dmead@lucigen.com
Small Business Information at Submission:

LUCIGEN CORPORATION
2120 W. GREENVIEW DR. SUITE 9 MIDDLETON, WI 53562

EIN/Tax ID: 391923306
DUNS: N/A
Number of Employees: N/A
Woman-Owned: No
Minority-Owned: No
HUBZone-Owned: No