Improved Ligation-Mediated PCR Method Coupled with T7 RNA Polymerase for Sensitive DNA Detection

摘要

The ligation-mediated polymerase chain reaction (PCR) method is widely applied for detecting short-length DNA target. The primary principle of this method is based on the linkage of two separated DNA probes as PCR templates via simultaneous hybridization with DNA target by DNA ligase. Even before taking into account low ligation efficiency, a 1:1 stoichiometric ratio between DNA target and the produced PCR template would put an intrinsic limitation on the detection sensitivity. In order to solve this problem, we have developed an improved ligation-mediated PCR method. It is designed such that a transcription reaction by T7 RNA polymerase is integrated into the ligation reaction. In this way, the produced joint DNA strand composed by two DNA probes can be used as a template both in the transcription reaction and the following PCR process. Then a great number of RNA strands containing the same sequence as DNA target are transcribed to act as a target to initiate new cyclic reactions of ligation and transcription. The results indicate that our proposed method can improve the detection sensitivity by ～2 orders of magnitude compared with the conventional ligation-mediated PCR method.