Detection of DNA Target with Highly Enhanced Specificity by Self-Circularization Rolling Circle Amplification

摘要

DNA detection is widely used for gene analysis. However, false-positive results are difficult to be avoided due to the non-specific amplification. Here, we described a novel RCA approach to improve the specificity. With the help of TspR Ⅰ (endonuclease), DNA was cleaved into smaller duplex fragments, including the target DNA fragment, with 9 nt sticky ends. A duplex adaptor with two sticky ends which were complementary to that of the target fragment was designed. After hybridization of the adaptor with the target fragments, T4 DNA ligase was used to ligate them to form a circular ds DNA with a gap. Then RCA was carried out from the free 3'-end of the open strand by Phi29 DNA polymerase with two primers which were complementary to the target sequence. Different from the traditional padlock-RCA, SC-RCA showed excellent specificity by amplifying the specific DNA target other than the added probe.