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.
The field of molecular diagnostics is a growing market with a current estimated value of $800 million. 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. ? ? ?
