Methods for insertion of nucleic acids into circular vectors

Abstract

The present invention provides vectors and methods which improve the efficiency of nucleic acid insertion into circular vectors, which generally facilitate nucleic acid cloning and specifically facilitate the preparation of DNA libraries. In general, the present invention involves separation of the cloning process into two distinct steps: (a) insertion which is done at a high nucleic acid concentration favoring intermolecular joining, and (b) circularization which is performed at a low nucleic acid concentration favoring intramolecular circularization. The present vectors generally have distinct insertion ends and circularization ends which are blocked from covalent joining during the insertion step. Circularization ends contemplated by the present invention include complementary cohesive ends and topoisomerase-linked ends. The present vectors and methods allow minute amounts of nucleic acid inserts to be efficiently cloned. Moreover, little or no insert size selection occurs with the present methods so that large as well as small nucleic acid inserts are readily inserted into the present vectors. Thus, DNA libraries which are representative of the entire range of size of DNA inserts can be made, and, for example, full length cDNA libraries are readily obtained.

1 - 32 . (Canceled)  
     
     
         33 . A mixture formed from a joining reaction of a population of linearized vectors with a population of nucleic acid fragments, wherein at least 95% of said nucleic acid fragments are inserted into circularized vectors and wherein at least 95% of said circularized vectors contain only one nucleic acid fragment insert.  
     
     
         34 . The mixture of  claim 33  wherein said population of nucleic acid fragments is cDNA or genomic DNA.  
     
     
         35 . A population of prokaryotic or eukaryotic host cells comprising the mixture of  claim 33 .  
     
     
         36 . A population of circularized vectors containing nucleic acid fragment inserts which is isolated from said population of prokaryotic or eukaryotic host cells of  claim 35 .  
     
     
         37 . A linearized vector comprising an origin of replication, an insertion site, and two complementary cohesive circularization ends, wherein: 
 each of said cohesive circularization ends is at least about 20 base pairs from said insertion site;    Said cohesive circularization ends are between about 8 and about 50 nucleotides in length; and    upon hybridization ligase does not substantially covalently join said cohesive circularization ends.    
     
     
         38 . The linearized vector of  claim 37  wherein said vector is cleaved in said insertion site with at least one restriction enzyme and dephosphorylated.  
     
     
         39 . (Canceled).  
     
     
         40 . A kit comprising a first compartment containing the linearized vector of any one of claims or  38 .  
     
     
         41 . The kit of  claim 40  which further comprises: 
 a second compartment containing a DNA ligase;    a third compartment containing a buffer comprising polyethylene glycol of high molecular weight; and    a fourth compartment containing a buffer comprising a salt.    
     
     
         42 - 50 . (Canceled).