Method for assembly of multiple DNA fragments

#The present application is a continuation of International Application No. PCT/IL98/00096, filed on Feb. 26, 1998, the disclosure of which is incorporated by reference herein.

#The present invention relates to the field of genetic engineering. More particularly, the invention relates to a novel method for assembling two or more DNA fragments with high efficiency and yield.

#Building DNA constructs is the basis of genetic engineering. Manipulating recombinant DNA and its incorporation into vectors is common knowledge in the art. For example, fragments obtained by PCR techniques, may be incorporated into plasmids for cloning purposes. Generating specific DNA constructs by joining together different DNA segments, each encoding specific properties and/or possessing specific functions, is also known in the art. The state of the art has been published in a very large number of books, articles, patent applications, patents and the like, and is usually readily available and known to all of skill in the art. For example, a comprehensive account of DNA cloning procedures is provided in the three volume text by Sambrook et al. (1989) entitled “Molecular Cloning—a Laboratory Manual” 2nd edition, Cold Spring Harbor Laboratory Press. This extensive account of the prior art techniques for the combination of DNA fragments, cloning and expression thereof, is included herein by reference, in its entirety.

#FIGS. 1-6 illustrate plasmids produced in Example 3. In the FIGS. 1-6, the dark, cross-hatched bar depicts the fragment amplified from pBR322 which carries the Ampr gene and the origin of replication. The empty bar depicts the fragment PCR amplified from pBR322 which carries the Tetr gene. The hatched bar depicts the PCR amplified fragment from pACYC184 which carries the Cmr gene. The arrows mark primers, where the arrow head is the 3′ end of the primer, and the wavy tails mark the various repetitive sequences at the 5′ end of the primers. The primer's number is given above or below the arrow. The black area denotes repetitive sequences situated at the connection between two fragments. FIG. 12 illustrates the plasmid produced in example 4. The empty, cross-hatched and hatched bars have the above meanings. The arrows mark the primers which are used for the seamless junction of the fragments.

#FIG. 1 is plasmid pGS101. There is an overlap of 12 bp between every two fragments which are joined together. The plasmid is assembled from three fragments.

#FIG. 2 is plasmid pGS102. There is an overlap of 12 bp between every two fragments which are joined together. The plasmid is assembled from four fragments.

#Overview

#The present invention is based on the surprising discovery that by increasing the length of complementary overhangs of two DNA fragments from 12 bases, as in the prior art, to 15 bases or more, the tendency of the fragments to be connected to each other rises, steeply increasing, from close to 0% when 12 base overhangs are used, to almost 100% when around 20 bases are employed. When long complementary overhangs of 15 bases or more are used, a large number of DNA fragments can be assembled in a single step in a predetermined, directional manner. In addition, when connections between just two fragments are made, the yield of cloned products is much higher than that of the prior art, when operating according to the invention.

#A number of procedures can be used in order to create these overhangs. In the examples detailed below, three different procedures were used.