Dna joining method

Abstract

The present invention provides a method to directionally clone any linear template DNA molecule into any linearized vector. The vector ends may be generated from any restriction enzyme cleavage. The method does not require a ligation step nor the use of carefully controlled conditions as is required with methods involving specific exonucleases alone. It has been determined that specific DNA polymerases are able to efficiently join one or more linear DNA molecules sharing ends with appropriate complementation.

1 - 30 . (canceled) 
     
     
         31 . A kit for direct cloning of a linear DNA molecule comprising, in separate containers, a DNA polymerase and a reaction buffer, wherein the DNA polymerase has intrinsic exonuclease activity and is capable of performing a DNA joining reaction. 
     
     
         32 . The kit according to  claim 31 , further comprising a stimulatory factor. 
     
     
         33 . The kit according to  claim 32 , wherein the stimulatory factor is a single strand DNA binding protein. 
     
     
         34 . The kit according to  claim 31 , further comprising reagents to perform a positive control reaction. 
     
     
         35 . The kit according to  claim 34 , wherein the reagents to perform a positive control comprise a linearized vector, an insert DNA with first and second ends having appropriate complementary sequences, a DNA polymerase having intrinsic exonuclease activity that is capable of performing a DNA joining reaction, and a reaction buffer. 
     
     
         36 . The kit according to  claim 35 , wherein the reagents to perform a positive control further comprise a stimulatory factor. 
     
     
         37 . The kit according to  claim 36 , wherein the insert DNA is a PCR-amplified product. 
     
     
         38 . A DNA molecule prepared using a method of joining two or more linear DNA molecules comprising:
 obtaining two or more linear DNA molecules, each having an x and x′ strand having opposite polarities, wherein the 5′ end of the x′ strand of each linear DNA molecule has a sequence of nucleotides that is complementary to the 5′ end of the x strand of the linear DNA molecule to which it is to be joined; and   incubating the two or more DNA molecules in the presence of a DNA polymerase under conditions whereby the two or more linear DNA molecules are joined;   wherein the DNA polymerase has intrinsic exonuclease activity and joins the DNA molecules.   
     
     
         39 - 42 . (canceled) 
     
     
         43 . A composition for cloning DNA molecules, comprising a carrier and DNA polymerase having exonuclease activity and capable of joining linear DNA molecules. 
     
     
         44 . A method of cloning DNA molecules comprising incubating DNA molecules in the presence of a DNA polymerase molecule having exonuclease activity and capable of joining DNA molecules. 
     
     
         45 . The method according to  claim 44 , wherein the DNA molecules are produced by PCR. 
     
     
         46 . The method according to  claim 44 , wherein the DNA polymerase comprises a polymerase selected from the group consisting of vaccinia virus DNA polymerase, T4 DNA polymerase and the Klenow fragment of  E. coli . DNA polymerase I. 
     
     
         47 . A method of circularizing linear DNA molecules for site directed mutagenesis comprising:
 PCR amplifying a linear DNA molecule in the presence of primers that will introduce substantially complementary nucleic acid sequences onto each end of the linear DNA molecule; and   incubating the PCR amplified linear DNA molecule in the presence of a DNA polymerase under conditions where the linear DNA molecule is circularized;   wherein the DNA polymerase has intrinsic exonuclease activity and circularizes the DNA molecule.