Development of nucleic acid detection method by utilizing flap endonuclease activity

Abstract

We herein describe novel strategies to detect various type of nucleic acid based on Flap endonuclease (FEN). FEN is a class of nucleolytic enzymes which hydrolyzes the phosphodiester bond in DNA backbone only when the target nucleic acid hybridizes with two specific nucleic acid probes. Since the FEN shows the high specificity and kinetics when it reacts with nucleic acid, it is widely utilized in molecular diagnostics including SNP genotyping and nucleic acid detection. Based on these characteristics, in chapter 1, we describe that FEN–initiated novel isothermal nucleic acid amplification strategy to overcome the limitation of the exponential amplification (EXPAR) and enzymatic repairing amplification (EAR), which were widely utilized in isothermal amplification of nucleic acid, enabling the detection of long target nucleic acid with high sensitivity. In chapter 2, we describe novel nucleic acid detection strategy based on FEN activity of Taq DNA polymerase combined with electrochemical analytical method which is considered as most promising technology against other optical method due to less expensive, easier to handle, and more portable than other methods. Finally, in chapter 3, we described a isothermal detection of target RNA based on FEN which cannot be conducted well in conventional FEN-based nucleic acid detection strategy. Since the FEN-based technologies described in this dissertation can be utilized for detecting all types of nucleic acid, it would be able to cause social or economic big ripple effects in the field of future in vitro diagnostic.