The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro. The reaction produces linear products, and as of yet, closed circular products have not been possible. Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells. Here, we describe an in vitro procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for sitedirected mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.The polymerase chain reaction (PCR) (1-3) is a powerful technique for in vitro amplification of nucleic acids. Although circular and linear nucleic acids can serve as templates for PCR, the resulting products have always been linear molecules. Until now, closed circular DNA, the replicatively competent form of many DNA molecules, could only be amplified in appropriate host cells. Here, we describe and demonstrate an in vitro procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. The essence of LDA is the inclusion of a thermostable DNA ligase in a PCR reaction that uses a closed circular DNA as template. After a primer is fully extended on the circular template, the ligase closes the gap to form a double-stranded DNA. Following thermal denaturation, the two circular DNA strands serve as templates for the next round of extension and ligation. Through thermal cycling, closed circular DNA is amplified exponentially.To demonstrate the feasibility of using LDA to generate and amplify closed circular DNA, two 5′ phosphorylated primers (16 and 17 nt long) were used to mutate and amplify a circular plasmid of 1990 bp. The primers are complementary to different strands of the plasmid in an inward orientation (Fig. 1A). One possesses a single G to A mismatch on an HphI site in the plasmid. The reaction mixture (50 µl) contained 10 ng of native plasmid, 10 pmol of each primer, 10 nmol of dNTPs, 5 nmol of ATP, 2.5 U Pfu DNA polymerase (Stratagene, La Jolla, CA), 4 U Pfu DNA ligase (Stratagene), in 1 cloned pfu DNA polymerase reaction buffer consisting of 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 1% Triton X-100 and 100 µg/ml BSA. The mixture was pre-incubated at 70_C for 10 min allowing the ligase to repair any nicks in the template. It was then subjected to thermal cycling at 95_C for 1 s (denaturation), 50_C for 1 s (annealing), 72_C for 4 min (extension), 95_C for 1 s (denaturation) and 72_C for 4 min (annealing, extension and ligation) for 20 cycles. As a control, a PCR was performed under similar conditions, except that the DNA ligase was not included. The reaction mixtures were analyzed directly by electrophoresis into 1% agarose gels followed by staining with ethid...
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