DNA ligases are more frequently being used as a tool in molecular biology applications that include nucleotide sequence detection, single nucleotide polymorphism (SNP) detection, protein detection, and """"""""next generation"""""""" sequencing by ligation. With the increased demand for DNA ligases in the field of biotechnology, so is the need for improved fidelity of ligation. Although many approaches to improving ligation fidelity have been employed, most involve use of ligases from different biological sources, point mutations of key amino acid residues, and modified reaction conditions. Herein, we propose a slightly different approach to improving the stringency of ligation, which employs a set of chemically modified ligation components. In our three-pronged approach, we propose the evaluation of chemically modified variants of the ATP cofactor, the donor probe, and the acceptor probe. The significance of this approach is great because each of these three components makes contacts with different key amino acid contacts within the ligase. It is hoped that subtle chemical alterations to the nucleic acid component of DNA ligase may in turn induce an improvement in the fidelity of ligation.

Public Health Relevance

The field of molecular diagnostics is a growing market with a current estimated value of $20.5 billion. One key class of enzymes that are used in these efforts is the DNA dependent DNA ligases. To further improve the accuracy of the DNA joining reaction catalyzed by DNA ligases, we propose the investigation of chemically modified components.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
5R44GM085860-03
Application #
8012837
Study Section
Special Emphasis Panel (ZRG1-IMST-E (15))
Program Officer
Maas, Stefan
Project Start
2008-07-07
Project End
2013-01-31
Budget Start
2011-02-01
Budget End
2013-01-31
Support Year
3
Fiscal Year
2011
Total Cost
$366,385
Indirect Cost
Name
Trilink Biotechnologies, Inc.
Department
Type
DUNS #
945720043
City
San Diego
State
CA
Country
United States
Zip Code
92121