Methods and compositions for DNA manipulation

#The present invention relates to compositions and methods for cloning and/or manipulating target DNA molecules at a desired sequence in a single experimental format.

#In the prior art, methods for manipulation and cloning of DNA include: amplification of DNA by Polymerase Chain Reaction (PCR); cleavage of DNA with restriction endonucleases; and ligation to create recombinant molecules. Limitations of these techniques include: lack of suitable restriction sites in DNA which creates experimental difficulties in accessing a desired location of the DNA sequence for a particular manipulation; poor yield of vector compatible molecules which arises from the template-independent terminal transferase activity of polymerase which introduces a non-template nucleotide at the 3′-termini of amplified products (Clark, Nucl. Acid. Res., 16:9677-9686 (1988)); and incompatibility of the termini of the amplified fragments with termini of recipient molecules thereby preventing efficient insertion of the fragments into vectors or fusion of two or more PCR products to one another. Addition of the 3′ nucleotide as a result of terminal transferase activity of the polymerase may be overcome by end polishing of PCR products using a polymerase with 3′ to 5′ exonuclease activity (Hemsley, et al., Nucl. Acid. Res., 17:6545-6551 (1989)). Alternatively, specially prepared vectors that carry 3′-T overhangs may be used to clone PCR products carrying non-template adenine at the 3′ ends (Marchuk, et al. Nucl. Acid. Res., 19:1154 (1990)). However, both the blunt-end insertion and the T/A overhang insertion are inefficient and the above methods do not permit control over the orientation of the inserted fragment in the vector.

#Efforts to improve the efficiency of blunt-end insertion of a DNA of interest by eliminating the background of self-ligated vector, include performing an insertion into a SmaI-linearized vector in the presence of SmaI restriction endonuclease which will cleave the self-ligated vector molecules at the re-generated SmaI sites (Liu, et al., BioTechniques, 12:28-30 (1992)). The PCR-Script Cloning Systems of Stratagene, Ltd. (La Jolla, Calif.) uses the rare-cleavage restriction enzyme SrfI for a similar purpose. The above methods are ineffective if the PCR product includes any internal SmaI or SrfI sites respectively, or if the site is re-generated after the insertion into the vector DNA.

#FIG. 1 shows how a 3′ single-stranded extension of desired length and composition is generated on the left-side of a cleaved polynucleotide molecule:

#(a) inserted into a polynucleotide molecule is a cassette, having boundaries (13), and containing site A recognized by nicking endonuclease A which nicks within site A on one strand as indicated by the arrow and site B located downstream of site A recognized by restriction endonuclease B, which cleaves both strands as indicated by the arrows;

#(b) the polynucleotide molecule containing the cassette showing breaks in the phosphodiester bond backbone from cleavage with nicking endonuclease A and restriction endonuclease B;

#Definitions

#The following terms as used in the Description and in the accompanying claims have been defined below. These definitions should be applied unless the context in which the terms are used requires otherwise.

#The term “single-stranded extension” as used herein refers to a single-stranded region extending from a double-stranded region of a polynucleotide molecule.