A simple rapid detection method of DNA based on ligation-mediated real-time fluorescence PCR

Abstract

Polymerase chain reaction (PCR) has been widely used for detecting long chain DNA or RNA of viruses, bacteria and cytokines, but it is difficult to detect DNA or RNA with short length sequences. In this work, we developed a simple and rapid detection method for short length DNA sequences in complicated matrices based on ligation-mediated PCR. Two probes, both designed as 52 bases and respectively partly complementary to the half-sequence of target DNA, could simultaneously hybridize to the target DNA, then to be ligated by T4 DNA ligase to form a long chain as PCR template for amplification. With the specific hybridization of the two probes and target DNA, and the PCR going on, a target with 16 bases was selectively detected with content as low as 200 fM, and the linear range spanned over five orders of magnitude. This method was successfully applied to the detection of target DNA in complicated biological samples such as cell lysate with satisfactory results.