Thermally Activatable Probes and Cofactors for Hot Start Ligase Chain Reaction

DESCRIPTION (provided by applicant):The Ligase Chain Reaction (LCR) is a DNA amplification technique that can either be utilized in conjunction with Polymerase Chain reaction (PCR), or as an alternative to PCR, due to its equal ability to support exponential signal amplification. LCR is being used as a tool in molecular biology and diagnostic applications, especially for nucleotide sequence detection and single nucleotide polymorphism (SNP) detection. However, performance problems and non-specificity issues, including the accumulation of false-positive signals, often make conventional LCR an unlikely choice in clinical diagnostics and in the development of ligation-based applications. Herein, we propose a novel Hot Start approach for LCR. Similar to the benefit seen for Hot Start activation methods in PCR, the Hot Start LCR approach is predicted to have substantially improved specificity and performance, compared to conventional LCR. Hot Start LCR employs chemically modified ligation components, including oligonucleotide probes and cofactors, containing thermally labile protecting groups for heat-triggered LCR. We believe that this approach will be a significant improvement to current methods because it has the high potential to replace a number of applications where traditional PCR, Gap-LCR or PCR/ligation combination approaches are either expensive, not applicable, or show problematic performance. PUBLIC HEALTH RELEVANCE: As of 2009, the molecular diagnostics market has an average annual growth rate of12- 15%, with an estimated value of 3-3.5 billion worldwide, where 2 to 2.5 billion of the market is in the USA. The Ligase Chain Reaction (LCR) is a substantial component of this market. We propose to improve the specificity of conventional LCR by developing a novel approach that includes Hot Start activation of LCR by using modified oligonucleotide probes and cofactors with thermolabile protecting groups.