Rapid and enzymeless cloning of nucleic acid fragments

Abstract

A method for cloning a nucleic acid fragment into a vector by flanking the fragment with first and second adapter sequences, and contacting the fragment with the vector having sequences homologous to the first and second adapter sequences under conditions such that the nucleic acid fragment is incorporated into the vector by homologous recombination in vivo in a host cell. Additionally, a method for selecting for a successful transformation of a vector by a nucleic acid insert. Also, systems for cloning a nucleic acid fragment into a vector without at least one of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, or other DNA modifying enzymes. Further, a kit for cloning a nucleic acid fragment into a vector.

1 - 43 . (canceled)  
     
     
         44 . A system for cloning a nucleic acid fragment into a vector without at least one of a restriction enzyme, a ligase, a gyrase, a topoisomerase, or a single stranded DNA binding protein, the system comprising a nucleic acid fragment flanked by first and second adapter sequences and a vector having sequences homologous to the first and second adapter sequences wherein the nucleic acid fragment is adapted to incorporate into the vector by recombination.  
     
     
         45 . The system of  claim 44 , wherein the nucleic acid fragment flanked by the first and the second adapter sequences is generated by PCR and without the use of at least one of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, a topoisomerase, or any other DNA modifying enzyme.  
     
     
         46 . The system of  claim 44 , wherein the nucleic acid fragment flanked by the first and the second adapter sequences is a transcriptionally active PCR fragment.  
     
     
         47 . The system of  claim 44 , wherein the recombination comprises homologous recombination.  
     
     
         48 . A system for cloning a nucleic acid fragment into a bacterium without the use of a restriction enzyme, a ligase, a gyrase, or a single stranded DNA binding protein, the system comprising a nucleic acid fragment flanked by first and second adapter sequences and a bacterium bearing a vector, the vector having sequences homologous to the first and second adapter sequences, wherein the nucleic acid fragment is adapted to incorporate into the vector within the bacterium by recombination.  
     
     
         49 . A kit for cloning a nucleic acid fragment into a vector comprising reagents for amplification of the nucleic acid fragment, wherein the reagents upon amplification provide for a nucleic acid fragment flanked by first and second adapter sequences, a vector, a competent cell, or a competent cell bearing the vector, and the competent cell is ready to be transformed by electroporation or chemical transformation.  
     
     
         50 - 74 . (canceled)