Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2002-01-14
2004-02-24
Leffers, Jr., Gerald G. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S067000, C435S252300, C435S320100, C435S471000, C435S256500, C435S476000, C435S091100, C435S091400, C536S023100
Reexamination Certificate
active
06696282
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a novel system and method for the sequential, directional cloning of multiple DNA sequences into a single vector.
2. Description of the Prior Art
The directional ligation of multiple DNA sequences within vectors is often hindered by the inability to force the orientation of subsequently ligated DNA fragments. This necessitates determination of fragment orientation following each ligation event to select recombinant plasmids with the inserts in the correct orientation (Potter, 1996, Biotechniques, 21:198-200). In addition, when attempting to clone a number of unrelated DNA fragments into a single host, the number of usable restriction sites declines rapidly, due to the presence of the sites in the insert DNA(s). Although it is sometimes possible to insert multiple genes into a single vector using a combination of available multi-cloning site (MCS) restriction sites (Jach et al., 1995, Plant Journal, 8:97-109; and Yamano et al., 1994, Biosci. Biotechnol. Biochem., 58:1112-1114), the process is often impractical. Moreover, the process is even more unreliable when attempting to directionally clone more than two genes into the vector.
SUMMARY OF THE INVENTION
We have now discovered a method which combines the use of polymerase chain reaction (PCR) or oligonucleotide linkers and restriction enzymes which cleave recognition site sequences that have internal degeneracy to allow the sequential, directional cloning of multiple DNA sequences into a DNA vector. In this invention, a plurality of unrelated DNA sequences may be directionally cloned within a single vector by adding onto the ends of the sequences, restriction-sites with specific sequences which are cleaved by corresponding restriction endonucleases which recognize degenerate or variable recognition sites and which generate cohesive ends upon cleavage. The compatibility (or ability to anneal) of the cohesive ends on different DNA sequences is controlled by the choice of the nucleotide sequence within the recognition sequences of the restriction endonucleases, allowing the DNA sequences to be inserted or joined in any desired orientation.
Generally, a recipient DNA (such as a vector) is provided which has a first restriction site having a degenerate recognition sequence with a predetermined nucleotide sequence, and which upon digestion with its restriction enzyme generates cohesive ends. A DNA sequence to be inserted into the recipient DNA is provided with a different restriction site at each end, which also include degenerate recognition sequences. However, the nucleotide sequences of these degenerate recognition sequences are selected such that upon digestion, they give rise to first and second cohesive ends which are each complementary to only one of the cohesive ends on the recipient DNA. The first cohesive end on the insert DNA is only complementary to one cohesive end on the recipient DNA, while the second cohesive end on the insert DNA is only complementary to the other cohesive end on the recipient DNA. Thus, the directionality or desired orientation of the ligation of the inserted DNA to the recipient DNA or vector is ensured. Furthermore, by choosing such restriction sites which are the same (cleaved by the same restriction enzyme), or different (cleaved by different enzymes), the user may selectively predetermine if the functional restriction site is or is not regenerated after ligation. When the cohesive ends generated from two of the same restriction sites are annealed, the functional restriction site will be regenerated. In contrast, the cohesive ends generated from two different restriction sites, although complementary, will not regenerate a functional restriction site when annealed.
These restriction sites may be selectively incorporated onto the end(s) of any DNA sequence of interest using PCR by adding the restriction sites onto the termini of the 5′ and/or 3′ primers, or by adding linkers to the DNA sequence.
In this process, the first DNA sequence of interest may be inserted into-the vector using either the process of this invention, or a variety of known techniques, including ligation into the vector at restriction sites generating blunt ends or cohesive ends, or a combination thereof. For instance, at least one end of the DNA sequence may be provided with a restriction site generating a cohesive end upon cleavage, which may then be inserted into the vector at any site which generates complementary cohesive ends.
To facilitate the ligation of additional DNA sequences, the first DNA sequence (further) includes one of the above-mentioned restriction sites having a degenerate recognition sequence adjacent (near) a selected end which also generates a cohesive end upon digestion with its corresponding restriction enzyme. This should be different from any other restriction sites present on the first DNA sequence, and should be unaffected by any initial restriction enzymes which may be used to insert the first sequence into the vector. This site should also be internal to any other different restriction sites used to insert the first DNA sequence into the vector to ensure that it is preserved.
The second DNA sequence of interest which is to be ligated adjacent to (upstream or downstream) from the first sequence is also provided with a restriction site adjacent to a selected first end that is different from the restriction site on the first sequence, and has a degenerate recognition sequence which, upon cleavage with its corresponding restriction enzyme, generates a cohesive end. The nucleotide sequences of the degenerate regions in these restriction sites (adjacent to the selected end of the first DNA sequence and the first end of the second DNA sequence) are selected such that the cohesive ends generated upon cleavage will be complementary to each other. If further DNA sequences are to be inserted into the vector adjacent to the second DNA sequence, the-second DNA sequence should also include an additional restriction site adjacent to its opposite or second end which is essentially the same as the above-mentioned restriction site on the first end of the first DNA sequence. Moreover, because the restriction sites at the ends of the second DNA sequence generate asymmetric cohesive ends when cleaved, the directionality or orientation of the insertion into the vector may be readily controlled by selection of the restriction sites and the nucleotide sequences of their degenerate internal recognition regions.
Upon digestion of the restriction sites on the second DNA sequence and the restriction site on the selected end of the first DNA sequence (now contained within the recombinant plasmid) with their restriction enzymes, each of the ends on the cut vector will be compatible to only one of the ends on the second DNA sequence, ensuring directionality. Specifically, the digestion of the restriction site on the first DNA sequence will generate overhangs on each end of the cut vector (one adjacent to the first DNA sequence and the other at the opposite end of the vector). The restriction site on the first end of the second DNA sequence will generate a cohesive end that is only complementary to the cohesive end adjacent to the first DNA sequence (i.e., at the selected end of the first DNA sequence), while the restriction site on the second end of the second DNA sequence will generate a cohesive end which is only complementary to the cohesive end on the opposite end of the cut vector. Upon ligation of these overhangs, not only will the second DNA sequence be inserted into the vector adjacent to the first DNA sequence in the desired orientation, but the restriction site at the second end of the second sequence will also be regenerated. Recreation of this restriction site will allow insertion of a further DNA sequence.
Any number of additional DNA sequences of interest may then be inserted into the vector sequentially from the second DNA sequence following the same protocol described for the second sequence.
In accordance with this disc
Hohn Thomas M.
Jones James D.
Leathers Timothy D.
Deck Randall E.
Fado John D.
Leffers, Jr. Gerald G.
The United States of America as represented by the Secretary of
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