The importance of primer to match the padlock DNA for synthesis of the circular DNA template was also investigated. In Fig. , the steps for synthesis of the circular DNA template are illustrated. A circularized DNA template can be formed only when the primer is fully complementary to pair with the padlock DNA. A mismatched ssDNA is not able to allow the T-4 DNA ligase to produce the circularized DNA template from the padlock DNA. Fig. shows the electrophoretic DNA bands from the padlock DNA, the primer, the circular DNA template, and the products of the rolling circle amplification using the circular DNA template. From the electrophoresis image, it is clear that the circular DNA template (Lane 3) and the non-circularized padlock DNA (Lane 4) display different mobility. The padlock DNA band in Lane 1 and the non-circularized padlock DNA band in Lane 4 have almost the same mobility, suggesting that the mismatched ssDNA was not able to give useful circular DNA template. In Lane 3 the circular DNA structure has a slower mobility than the padlock DNA (Lane 1). Lane 5 and 6 are the products obtained by RCA using the non-circularized padlock DNA and circular DNA template. Compared to RCA using the noncircularized padlock DNA that produced heterogeneous products (Lane 5), the circular DNA template produced only high molecular weight products represented by an intense band on the top of the gel image (Lane 6). The impact of reaction time of RCA was also studied. Samples were taken from the RCA reaction mixture at 0, 2 and 20 h and analyzed by gel electrophoresis. As shown in Fig. , before RCA the reaction mixture showed multiple DNA bands of varying lengths. After 2 h of RCA, the product generated one intense band on the top of the gel. The 20 h RCA produced predominantly long strand DNA that appeared as a dark band on the top of the gel. Based on these results, we conclude that the designed circular DNA template and the RCA reaction were successful.