Unrestricted mutagenesis and cloning method

Abstract

The invention relates to methods for amplifying, modifying, mutating and cloning DNA of any size. These methods comprise a series of PCR reactions, which are punctuated by ligation reactions.

1 - 40 . (canceled) 
     
     
         41 . A kit comprising:
 a package insert providing instructions to carry out the methods of claim  1 ;   a DNA polymerase;   a DNA polymerase buffer;   a DNA ligase;   a ligase buffer;   a control template;   control Starter Primers; and   control Opener Primers.   
     
     
         42 . The kit of  claim 41  further comprising a reverse transcriptase and a reverse transcriptase buffer. 
     
     
         43 . A method of modifying a nucleic acid comprising the steps of:
 (a) contacting an Original DNA, comprising a Target DNA Fragment containing a Modification Target that is flanked by two restriction sites, with a DNA polymerase and two OIL Starter Primers, selected to amplify the target DNA fragment, so that the OIL Starter Primers anneal to segments of the Original DNA at the ends of the Target DNA Fragment;   (b) amplifying a portion of the Original DNA comprising the Target DNA fragment using polymerase chain reaction (PCR) with the OIL Starter Primers to generate a Starter DNA;   (c) contacting the Starter DNA with a DNA polymerase and two Opener Primers, selected to generate a modification in the Target DNA Fragment, so that the Opener Primers anneal to opposite strands of the adjacent segments of the Target DNA Fragment;   (d) amplifying the Starter DNA using PCR with the OIL Starter Primers and the Opener Primers to generate two PCR products whose 3′ ends anneal and act as primers to complete the intermediate DNA;   (e) self-ligating the intermediate DNA with DNA ligase to generate a Closed intermediate DNA; and optionally,   (f) contacting the Closed Intermediate DNA with the OIL Primers and a DNA polymerase to amplify the Closed Intermediate DNA and generate a Linear Modified DNA containing the modification.   
     
     
         44 . (canceled) 
     
     
         45 . A method of modifying a nucleic acid comprising the steps of:
 a) contacting in a first reaction mixture an Original DNA, comprising a Target DNA Fragment containing a Modification Target flanked by restriction sites, with OIL Starter Primer A and Opener Primer B;   b) amplifying a portion of the Original DNA comprising the Target DNA fragment in the first reaction mixture to generate a portion of a first intermediate DNA;   c) contacting in a second reaction mixture the Original DNA with OIL Starter Primer B and Opener Primer A1   d) amplifying a portion of the Original DNA comprising the Target DNA fragment in the second reaction mixture to generate a portion of a second intermediate DNA;   e) combining and extending the first and second Intermediate DNAs to generate a third intermediate DNA, which may be optionally amplified   f) with Opener Primers A and B;   g) self-ligating the third intermediate DNA with DNA ligase to generate a Closed intermediate DNA; and optionally,   h) contacting the Closed Intermediate DNA with the OIL Primers and a DNA polymerase to amplify the Closed Intermediate DNA with the OIL Primers and a DNA polymerase to amplify the Closed Intermediate DNA and generate a Linear Modified DNA containing the modification, wherein the OIL Primers anneal to segments of the Original DNA at the ends of the Target DNA Fragment and wherein the OIL Primers are designed so that the Target DNA Fragment is amplified.   
     
     
         46 . The method of  claim 43 , wherein:
 At least one of the amplifying steps is accomplished by polymerase chain reaction (PCR).   
     
     
         47 . The method of  claim 43 , wherein:
 At least one of the primers is modified by phosphorylation at the 5′ end.   
     
     
         48 . The method of  claim 43 , further comprising the steps of:
 generating cohesive ends in the Liner Modified DNA and the Original DNA, by enzymes having exonuclease activity, so that the overhanging ends of the product of  claim 43  contains regions that are complementary to those of the Original DNA;   annealing the resulting products; and   ligating by DNA ligase.