Single-step single-molecule PCR of DNA with a homo-priming sequence using a single primer and hot-startable DNA polymerase

We have previously reported that a protein library can be constructed by di rectly combining PCR amplification of a single DNA molecule and cell-free p rotein synthesis. To specifically amplify single DNA molecules, however, tw o-step PCR with nested primers was used. Here we describe a simpler method for single-step amplification of a single molecule. The method involves the use of both hot-startable DNA polymerase and a DNA template that has homo- priming sequences at both ends for amplification using a single primer. The se two modifications greatly decreased the possibility of formation and sub sequent accumulation, respectively, of primer-dimers that inhibit the ampli fication of target template. In addition, a high-fidelity DNA polymerase wa s successfully used, resulting in the significant reduction of the accumula tion of mutations during amplification.