Primer-mediated polynucleotide synthesis and manipulation techniques

摘要

The invention provides improved techniques for conveniently manipulating polynucleotides of interest without the need to rely upon the presence of naturally occurring restriction sites. Additionally, using the methods and primers of the invention, one may synthesize a polynucleotide of interest in a form which is easily and directionally cloned into a DNA sequence of choice without necessarily introducing extraneous nucleotides in the final polynucleotide product. The methods of the invention employ releasable primers that comprise a recognition site for a releasing enzyme joined to a region for annealing to the polynucleotide template of interest. Polynucleotide sequences of interest are synthesized using one or more synthesis primers, wherein at least one of the primers is a releasable primer. After synthesis, the synthesis product is cleaved by a releasing enzyme. In a preferred embodiment of the invention, inhibitory base analogs are incorporated in the synthesis product to protect against the formation of unwanted internal cleavage products. In another embodiment of the invention, at least one of the releasable primers is bound to an immobilizing solid phase support so as to produce immobilized synthesis products that may be conveniently released by a releasing enzyme. Another aspect of the invention is to provide releasable primers and kits for performing the subject methods. Typically, such kits may comprise a releasing enzyme and one or more reagents for performing a polynucleotide synthesis reaction, preferably a cyclic amplification reaction.