Methods and compositions for cloning nucleic acid molecules

Abstract

The present invention is directed generally to methods facilitating the cloning of nucleic acid molecules. In particular, the invention relates to the use of polymerase inhibitors, including but not limited to anti-polymerase antibodies (such as anti-Taq antibodies) and fragments thereof, to inactivate residual polymerase activity remaining after the amplification (particularly via PCR) of a target nucleic acid molecule. The invention further provides compositions, particularly storage-stable compositions, comprising one or more components, such as one or more restriction endonucleases and one or more polymerase inhibitors, that are useful in cloning amplified or synthesized nucleic acid molecules by the above-described methods. The invention also relates to nucleic acid molecules produced by these methods, and to genetic constructs (such as vectors) and host cells comprising these nucleic acid molecules.

1 - 52 . (canceled) 
     
     
         53 . A method for cloning one or more nucleic acid molecules into one or more vectors comprising:
 (a) forming a mixture comprising said nucleic acid molecules to be cloned, one or more polymerase inhibitors and one or more restriction endonucleases; and   (b) ligating said nucleic acid molecules into one or more vectors to form one or more genetic constructs.   
     
     
         54 . the method of  claim 53 , wherein the mixture further comprises one or more polypeptides having polymerase activity. 
     
     
         55 . The method of  claim 53 , wherein the nucleic acid molecules to be cloned are amplified or synthesized nucleic acid molecules. 
     
     
         56 . The method of  claim 53 , further comprising transforming the one or more genetic constructs into one or more host cells. 
     
     
         57 . The method of  claim 53 , wherein the inhibitors and the restriction endonucleases are added simultaneously. 
     
     
         58 . The method of  claim 53 , wherein the inhibitors and the restriction endonucleases are added sequentially. 
     
     
         59 . A method of ligating and amplified or synthesized nucleic acid molecule into a vector with increased efficiency, comprising:
 (a) forming a mixture comprising the amplified or synthesized nucleic acid molecules and one or more polymerase inhibitors; and   (b) ligating the nucleic acid molecules into one or more vectors to form one or more genetic constructs.   
     
     
         60 . The method of  claim 59 , wherein the mixture further comprises one or more polypeptides having polymerase activity. 
     
     
         61 . The method of  claim 59 , further comprising transforming the one or more genetic constructs into one or more host cells. 
     
     
         62 . A composition comprising one or more restriction endonucleases and one or more polymerase inhibitors. 
     
     
         63 . The composition of  claim 62  wherein the one or more inhibitors are selected from the group consisting of an antibody or a fragment thereof, a chemical compound, an antibiotic, a heavy metal, an acid, a metal chelator, a nucleotide analogue, a sulfhydryl reagent, an anionic detergent, a polyanion, captan((N-[trichloromethyl]-thio)-4-cyclohexene-1,2-dicarboximide), an acidic polysaccharide, a binding protein or peptide, and combinations thereof. 
     
     
         64 . The composition of  claim 63 , wherein the inhibitor is an antibody or fragment thereof. 
     
     
         65 . The composition of  claim 64 , wherein the antibody is selected from the group consisting of an anti-Taq antibody, an anti-Tne antibody, an anti-Tma antibody, an anti-Pfu antibody and fragments thereof. 
     
     
         66 . The composition of  claim 62 , further comprising one or more polypeptides having polymerase activity. 
     
     
         67 . The composition of  claim 62 , further comprising one or more nucleic acid molecules.