Primer-mediated polynucleotide synthesis and manipulation techniques

Abstract

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.

What is claimed is:  
     
         1 . A method of producing a polynucleotide of interest, said method comprising the steps: 
 synthesizing the polynucleotide of interest in a polynucleotide synthesis reaction employing a first releasable primer, whereby a polynucleotide synthesis product is produced; and    cleaving the polynucleotide synthesis product with a releasing enzyme specific for the releasable primer, whereby a released synthesis product is produced.    
     
     
         2 . A method according to  claim 1 , wherein said polynucleotide synthesis reaction employs a second primer.  
     
     
         3 . A method according to  claim 2 , wherein the second primer is a releasable primer.  
     
     
         4 . A method according to  claim 2 , wherein said polynucleotide synthesis reaction is a cyclic amplification reaction.  
     
     
         5 . A method according to  claim 4 , wherein the cyclic amplification reaction is a polymerase chain reaction.  
     
     
         6 . A method according to  claim 1 , wherein the releasable primer comprises a recognition site for a type IIS restriction endonuclease.  
     
     
         7 . A method according to  claim 3 , wherein the first and second releasable primers comprise recognition sites for a type IIS restriction endonuclease.  
     
     
         8 . A method according to  claim 1 , wherein the releasable primer is bound to a solid phase support.  
     
     
         9 . A method according to  claim 1 , wherein at least one inhibitory base analog is present in the released synthesis product.  
     
     
         10 . The method according to  claim 9  wherein the inhibitory base analog is a methylated analog.  
     
     
         11 . The method according to  claim 10 , wherein the inhibitory base analog is 5-methyl-cytosine.  
     
     
         12 . The method according to  claim 11 , wherein the releasing enzyme is Eam1104I.  
     
     
         13 . A method according to  claim 3 , wherein at least one inhibitory base analog is present in the released synthesis product.  
     
     
         14 . A method according to  claim 13 , wherein the inhibitory base analog is a methylated analog.  
     
     
         15 . A method according to  claim 14 , wherein the inhibitory base analog is 5-methyl-cytosine.  
     
     
         16 . A method according to  claim 15 , wherein the releasing enzyme is Eam1104I.  
     
     
         17 . A method of constructing a polynucleotide of interest, said method comprising the steps of: 
 synthesizing a first polynucleotide of interest in a polynucleotide synthesis reaction employing first and second primers, wherein at least one of said first and second primers is a releasable primer, whereby a first polynucleotide synthesis is produced;    cleaving the first polynucleotide synthesis product with a releasing enzyme specific for a recognition site of said first releasable primer, whereby a first released synthesis product is produced;    synthesizing a second polynucleotide of interest in a second polynucleotide synthesis reaction employing third and fourth primers, wherein at least one of said third and fourth primers is a releasable primer, whereby a second polynucleotide synthesis product is produced;    cleaving the second polynucleotide synthesis product with said releasing enzyme specific for the recognition site of said third releasable primer, whereby a second released synthesis product is produced; and    ligating the first released synthesis product to the second released synthesis product.    
     
     
         18 . A method according to  claim 17 , wherein said first polynucleotide synthesis reaction is a cyclic amplification reaction.  
     
     
         19 . A method according to  claim 18 , wherein said second polynucleotide synthesis reaction is a cyclic amplification reaction.  
     
     
         20 . A method according to  claim 19 , wherein the first, second, third, and fourth primers are releasable primers.  
     
     
         21 . A method according to  claim 20 , wherein at least one inhibitory base analog is present in the first polynucleotide synthesis product.  
     
     
         22 . A method according to  claim 21 , wherein at least one inhibitory base analog is present in the second polynucleotide synthesis product.  
     
     
         23 . The method according to  claim 21  wherein the inhibitory base analog is a methylated analog.  
     
     
         24 . The method according to  claim 23 , wherein the inhibitory base analog is 5-methyl-cytosine.  
     
     
         25 . The method according to  claim 24 , wherein the releasing enzyme is Eam1104I.  
     
     
         26 . A releasable oligonucleotide primer, said primer comprising a recognition site for a releasing enzyme and an annealing region, wherein the releasing enzyme cleaves polynucleotide substrates at a site separate from the recognition site.  
     
     
         27 . A releasable oligonucleotide primer according to  claim 26 , wherein said releasing enzyme recognition site is a restriction endonuclease recognition site.  
     
     
         28 . A releasable oligonucleotide primer according to  claim 27 , further comprising nucleotides located 5′ and adjacent to the restriction endonuclease recognition site.  
     
     
         29 . A releasable oligonucleotide primer according to  claim 28 , wherein the restriction endonuclease recognition site is recognized by Eam1104I.  
     
     
         30 . A releasable oligonucleotide primer according to  claim 27 , wherein the primer is bound to a solid phase support.  
     
     
         31 . A set of releasable synthesis primers for seamless domain replacement comprising the first, second, third, and fourth releasable primers of the method of  claim 20 .  
     
     
         32 . A kit for seamless polynucleotide synthesis, said kit comprising a releasing enzyme.  
     
     
         33 . A kit according to  claim 32 , said kit further comprising an inhibitory base analog nucleoside triphosphate.  
     
     
         34 . A kit according to  claim 32 , said kit further comprising a thermostable DNA polymerase.  
     
     
         35 . A kit according to  claim 33 , wherein the releasing enzyme is a type IIS restriction endonuclease.  
     
     
         36 . A kit according to  claim 35 , said kit comprising 5-methylcytosine triphosphate and Eam1104I.