Methods for Nucleic Acid Manipulation

Abstract

A method for replicating and amplifying a target nucleic acid sequence is described. A method of the invention involves the formation of a recombination intermediate without the prior denaturing of a nucleic acid duplex through the use of a recombination factor. The recombination intermediate is treated with a high fidelity polymerase to permit the replication and amplification of the target nucleic acid sequence. In preferred embodiments, the polymerase comprises a polymerase holoenzyme. In further preferred embodiments, the recombination factor is bacteriophage T4 UvsX protein or homologs from other species, and the polymerase holoenzyme comprises a polymerase enzyme, a clamp protein and a clamp loader protein, derived from viral, bacteriophage, prokaryotic, archaebacterial, or eukaryotic systems.

1 .- 22 . (canceled) 
     
     
         23 . A process for generating a library of recombinant deoxyribonucleic acid sequences comprising:
 (a) combining a single-stranded deoxyribonucleic acid with a UvsX recombination factor to form a pre-treated single-stranded deoxyribonucleic acid;   (b) combining the pre-treated single stranded deoxyribonucleic acid with a double-stranded deoxyribonucleic acid duplex and a helicase to form a triple-stranded Holliday junction wherein the pre-treated single stranded deoxyribonucleic acid invades the double-stranded deoxyribonucleic acid duplex and pairs with one of the strands of the duplex and wherein the remaining unpaired other strand of the duplex forms an adjacent unpaired D-loop; and   (c) combining the triple-stranded Holliday junction with an endonuclease that specifically recognizes Holliday junctions to remove the unpaired D-loop and a ligase to form heterologous double-stranded deoxyribonucleic acids.   
     
     
         24 . The process of claim  1  wherein the helicase comprises bacteriophage T4 gene products 41 and 59. 
     
     
         25 . The process of claim  1  wherein the helicase comprises bacteriophage T4 UvsW. 
     
     
         26 . The process of claim  1  wherein the endonuclease comprises bacteriophage T4 gene product 49. 
     
     
         27 . The process of claim  1  further comprising combining a UvsY accessory factor with the UvsX recombination factor and single-stranded deoxyribonucleic acid. 
     
     
         28 . A process for generating a library of recombinant deoxyribonucleic acid sequences comprising:
 (a) combining a single-stranded deoxyribonucleic acid with a UvsX recombination factor and a double-stranded deoxyribonucleic acid duplex and a helicase to form a triple-stranded Holliday junction wherein the pre-treated single stranded deoxyribonucleic acid invades the double-stranded deoxyribonucleic acid duplex and pairs with one of the strands of the duplex and wherein the remaining unpaired other strand of the duplex forms an adjacent unpaired D-loop; and   (b) combining the triple-stranded Holliday junction with an endonuclease that specifically recognizes Holliday junctions to remove the unpaired D-loop and a ligase to form heterologous double-stranded deoxyribonucleic acids of different sizes.   
     
     
         29 . The process of claim  1  wherein the helicase comprises bacteriophage T4 gene products 41 and 59. 
     
     
         30 . The process of claim  1  wherein the helicase comprises bacteriophage T4 UvsW. 
     
     
         31 . The process of claim  1  wherein the endonuclease comprises bacteriophage T4 gene product 49. 
     
     
         32 . The process of claim  1  further comprising combining a UvsY accessory factor with the UvsX recombination factor and single-stranded deoxyribonucleic acid. 
     
     
         33 . A process for generating a library of recombinant deoxyribonucleic acid sequences comprising:
 (a) incubating a first double-stranded nucleic acid with an enzyme having exonuclease activity to form a plurality of single-stranded DNA regions having random sizes;   (b) combining the plurality of single-stranded DNA regions having random sizes with a UvsX recombination factor to form plurality of pre-treated single-stranded DNA regions;   (c) adding a second double-stranded nucleic acid to the plurality of pre-treated single-stranded DNA regions to form a plurality of three stranded crossover junctions;   (d) combining the plurality of three stranded crossover junctions with a helicase to form a plurality of Holliday junctions; and   (e) incubating the plurality of Holliday junctions with an endonuclease.   
     
     
         34 . The process of claim  1  wherein the helicase comprises bacteriophage T4 gene products 41 and 59. 
     
     
         35 . The process of claim  1  wherein the helicase comprises bacteriophage T4 UvsW. 
     
     
         36 . The process of claim  1  wherein the endonuclease comprises bacteriophage T4 gene product 49.