Ramification amplification: A novel isothermal DNA amplification method

We have developed a novel isothermal DNA amplification method with an ampli fication mechanism quite different from conventional PCR. This method uses a specially designed circular probe (C-probe) in which the 3 ' and 5 ' ends are brought together in juxtaposition by hybridization to a target. The tw o ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle. In the presence of an excess of prim ers (forward and reverse primers), a DNA polymerase extends the bound forwa rd primer along the C-probe and displaces the downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to the "rolling circle " replication of bacteriophages in vivo. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displa ce downstream DNA, generating a large ramified (branching) DNA complex. Thi s ramification process continues until all ssDNAs become double-stranded, r esulting in an exponential amplification that distinguishes itself from the previously described nonexponential rolling circle amplification. In this report, we prove the principle of ramification amplification. By using a un ique bacteriophage DNA polymerase, empty-set29 DNA Polymerase, that has an intrinsic high processivity, we are able to achieve significant amplificati on within 1 hour at 35 degreesC. In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.