Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1996-09-13
2001-07-17
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S006120, C530S350000
Reexamination Certificate
active
06261797
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of polynucleotide manipulation techniques, particularly amplification and cloning techniques.
BACKGROUND OF THE INVENTION
A significant problem with many of the currently available molecular biology techniques is their reliance upon naturally occurring convenient restriction sites. Modifications of the polymerase chain reaction (PCR) and other similar amplification techniques have been developed in an attempt to overcome this problem. In the absence of naturally occurring convenient restriction sites, it is possible to introduce restriction sites into the sequence of interest by using primers and PCR. However, this technique results in the presence of extraneous polynucleotides in the amplification products even after restriction digestion. Such extraneous polynucleotides can be problematic. For example, the introduction of unwanted nucleotides often imposes design limitations on the cloned product which may interfere with the structure and function of the desired gene products.
One method of joining DNA without introducing extraneous bases or relying on the presence of restriction sites is splice overlap extension (SOE). Yon et al., 1989,
Nucl. Acids Res.
17:4895. Horton et al., 1989,
Gene
77:61-68. This method is based on the hybridization of homologous 3′ single-stranded overhangs to prime synthesis of DNA using each complementary strand as template. Although this technique can join fragments without introducing extraneous nucleotides (in other words, seamlessly), it does not permit the easy insertion of a DNA segment into a specific location when seamless junctions at both ends of the segment are required. Nor does this technique function to join fragments with a vector. Ligation with a vector must be subsequently performed by incorporating restriction sites onto the termini of the final SOE fragment. Finally, this technique is particularly awkward when trying to exchange polynucleotides encoding various domains or mutation sites between genetic constructs encoding related proteins.
Another commonly used genetic manipulation technique is immobilized amplification, e.g., immobilized PCR. In techniques involving immobilized PCR, i.e., bound PCR, polynucleotide amplification products are immobilized on a solid phase support. Immobilization is typically accomplished through the use of streptavidin (or avidin) and biotinylated polynucleotides, antibody-hapten binding interactions, or through the covalent attachment of nucleic acids to solid supports. A serious limitation, however, of such conventional immobilization techniques is that the amplification products cannot be conveniently unbound from the solid phase support for use in subsequent manipulations, e.g., sequencing of the amplification products.
An additional problem with conventional techniques, particularly the manipulation of amplification reaction products, is that cleavage at certain restriction sites must be avoided in order to obtain desired polynucleotides. Presently, however, this can only be accomplished by cumbersome techniques such as partial digestions and methylase protection.
Accordingly, in view of the foregoing limitations of current recombinant DNA technology, it is of interest to provide improved techniques for conveniently manipulating polynucleotides without having to rely on naturally occurring convenient restriction sites. It is also of interest to provide methods of synthesizing polynucleotides in which some or all of the nucleotides introduced through synthesis primers may be conveniently removed from the final synthesis product. Additionally, it is of interest to provide improved methods of manipulating polynucleotide synthesis products by restriction enzymes which overcome the problems of cleavage at internal sites within the synthesis products. Further, it is of interest to provide an improved method of releasing amplification products that are bound to a solid phase support. The present invention meets these needs.
SUMMARY OF THE INVENTION
The present invention relates to improved methods of synthesizing polynucleotides of interest. The invention is based, in part, on the use of enzymes, referred to herein as releasing enzymes, which cleave polynucleotide substrates. In one embodiment of the invention, it is preferred that the site cleaved by the releasing enzyme is different or distal from the enzyme recognition site on the substrate. The methods of the invention employ primers which comprise a recognition site for a releasing enzyme joined to a region for annealing to the polynucleotide template of interest. These primers are referred to as releasable primers. Preferably, the recognition site for the releasing enzyme is joined 5′ to the annealing region.
In one embodiment of the invention, the releasable primers comprise a recognition site for a type IIS restriction endonuclease. The type IIS restriction endonuclease recognizes this site, but then cleaves the DNA in a sequence independent manner several base pairs 3′ to the recognition site. Optionally, releasable primers of the invention comprise additional nucleotides located 5′ and adjacent to the recognition site.
The releasable primers may be used for priming polynucleotide synthesis reactions, including, but not limited to, polymerase chain reactions and other amplification reactions.
The methods of the invention comprise the steps of synthesizing a polynucleotide sequence of interest with at least one releasable primer. The polynucleotide synthesis reaction may be, but is not necessarily, a cyclic amplification reaction. When polynucleotide synthesis occurs in a cyclic amplification reaction, the polymerase chain reaction (PCR) is particularly preferred for use. After synthesis, the synthesis product is cleaved by a releasing enzyme capable of recognizing the recognition site on the releasable primer. Restriction endonuclease inhibitory base analogs may be incorporated in the synthesis product to protect against unwanted cleavage of internal recognition sites by the releasing enzyme, yet still permit cleavage of the desired sites introduced by the releasable primer or primers.
In another embodiment of the invention, i.e., seamless domain replacement, a first synthesis product is produced using a pair of primers and a second synthesis product is produced using a second pair of primers, wherein at least one member of each pair of primers is a releasable primer. Both first and second synthesis products are subsequently cleaved by releasing enzymes. The resultant released first and second synthesis products may then be ligated to one another so as to produce a recombinant DNA construct that does not contain extraneous nucleotides introduced by the synthesis primers. This method may be used to conveniently replace one segment of a genetic construct with a similar (but different) segment of a second genetic construct.
In another embodiment of the invention, at least one releasable primer is bound to a solid phase support. After synthesis of a polynucleotide of interest using the bound primer, an immobilized synthesis product is produced. The immobilized synthesis product may be released by means of a releasing enzyme. Restriction endonuclease inhibitory base analogs may be incorporated in the synthesis product to protect against unwanted cleavage of internal restriction sites by the selected releasing enzyme, yet still permit cleavage of the desired restriction sites introduced by the releasable primer(s).
Another aspect of the invention is to provide releasable primers and kits for performing the subject methods. Typically, such kits comprise a releasing enzyme and one or more reagents for performing polynucleotide synthesis, e.g., a cyclic amplification reaction. Optionally, such kits further comprise nucleotide base analogs capable of inhibiting or substantially inhibiting cleavage by the releasing enzyme. Preferably, such inhibitory nucleotide base analogs are in the form of nucleoside triphosphates. The kits may also optionally comprise a polynucleotide
Padgett Kerstien A.
Sorge Joseph A.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Stratagene
Whisenant Ethan
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