Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-24
2004-12-07
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S462000, C435S476000, C435S477000, C435S252330, C435S196000
Reexamination Certificate
active
06828093
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to recombinant DNA technology. In particular, the invention relates to compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro.
BACKGROUND OF THE INVENTION
Molecular biotechnology has revolutionized the production of protein and polypeptide compounds of pharmacological importance. The advent of recombinant DNA technology permitted for the first time the production of proteins on a large scale in a recombinant host cell rather than by the laborious and expensive isolation of the protein from tissues which may only contain minute quantities of the desired protein (e.g., isolation of human growth hormone from cadaver pituitary). The production of proteins, including human proteins, on a large scale in a heterologous host requires the ability to express the protein of interest in the heterologous host. This process typically involves isolation or cloning of the gene encoding the protein of interest followed by transfer of the coding region into an expression vector that contains elements (e.g., promoters) which direct the expression of the desired protein in the heterologous host cell. The most commonly used means of transferring or subcloning a coding region into an expression vector involves the in vitro use of restriction endonucleases and DNA ligases. Restriction endonucleases are enzymes which generally recognize and cleave a specific DNA sequence in a double-stranded DNA molecule. Restriction enzymes are used to excise the coding region from the cloning vector and the excised DNA fragment is then joined using DNA ligase to a suitably cleaved expression vector in such a manner that a functional protein may be expressed.
The ability to transfer the desired coding region to an expression vector is often limited by the availability or suitability of restriction enzyme recognition sites. Often multiple restriction enzymes must be employed for the removal of the desired coding region and the reaction conditions used for each enzyme may differ such that it is necessary to perform the excision reactions in separate steps. In addition, it may be necessary to remove a particular enzyme used in an initial restriction enzyme reaction prior to completing all restriction enzyme digestions; this requires a time-consuming purification of the subcloning intermediate. Ideal methods for the subcloning of DNA molecules would permit the rapid transfer of the target DNA molecule from one vector to another in vitro or in vivo without the need to rely upon restriction enzyme digestions.
SUMMARY OF THE INVENTION
The present invention provides reagents and methods which comprise a system for the rapid subcloning of nucleic acid sequences in vivo and in vitro without the need to use restriction enzymes.
The present invention provides a method for the recombination of nucleic acid constructs, comprising: providing a first nucleic acid construct comprising, in operable order, an origin of replication, a first sequence-specific recombinase target site, and a nucleic acid of interest, a second nucleic acid construct comprising, in operable order, an origin of replication, a regulatory element and a second sequence-specific recombinase target site adjacent to and downstream from the regulatory element, and a site-specific recombinase; contacting the first and the second nucleic acid constructs with the site-specific recombinase under conditions such that the first and second nucleic acid constructs are recombined to form a third nucleic acid construct, wherein the nucleic acid of interest is operably linked to the regulatory element. The present invention contemplates the use of any type of regulatory element. In some embodiments of the present invention, the regulatory element comprises a promoter element, a fusion peptide (e.g., an affinity domain), or an epitope tag. In preferred embodiments, the nucleic acid of interest comprises a gene.
In some embodiments, the first nucleic acid construct further comprises a selectable marker.- In other embodiments, the second nucleic acid construct further comprises a selectable marker. The present invention contemplates that the first and second nucleic acid constructs both comprise selectable markers. In preferred embodiments the selectable markers of the first and second nucleic acid constructs are different from one another. Selectable markers include, but are not limited to a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, the aadA gene, the &PHgr;X174 E gene, the strA gene, and the sacB gene.
In preferred embodiments, the first nucleic acid construct further comprises a prokaryotic termination sequence. Prokaryotic termination sequences include, but are en not limited to the T7 termination sequence. In other preferred embodiments, the first nucleic acid construct further comprises a eukaryotic polyadenylation sequence. Polyadenylation sequences include, but are not limited to, the bovine growth hormone polyadenylation sequence, the simian virus 40 polyadenylation sequence, and the Herpes Simplex virus thymidine kinase polyadenylation sequence. In yet other preferred embodiments, the first nucleic acid construct further comprises a conditional origin of replication.
In preferred embodiments of the present invention, the first and second sequence-specific recombinase target sites are selected from the group consisting of loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, lox&Dgr;86, lox&Dgr;117, frt, dif, loxH and att. The present invention contemplates that the first and second sequence-specific recombinase target sites may comprise the same sequence or may comprise different sequences.
In yet other embodiments of the present invention, the first nucleic acid construct further comprises a polylinker.
The present invention contemplates that the recombination methods can be used in vitro and in vivo. In some in vivo embodiments, the site-specific recombinase is provided by a host cell expressing the site-specific recombinase. In some in vivo methods, the contacting of the first and the second nucleic acid constructs with the site-specific recombinase comprises introducing the first and said second nucleic acid constructs into a host cell under conditions such that the third nucleic acid construct is capable of replicating in the host cell.
The present invention further provides methods for precise transfer of nucleic acid molecules by recombination. In some embodiments, the first nucleic acid construct further comprises a third sequence-specific recombinase target site and said second nucleic acid constructs further comprises a fourth sequence-specific recombinase target site. In preferred embodiments, the first sequence-specific recombinase and the third sequence-specific recombinase in the first nucleic acid construct are located on opposite sides of the nucleic acid of interest. It is contemplated that the first and third sequence-specific recombinase target sites are contiguous with, adjacent to, or distant from the nucleic acid of interest. In particularly preferred embodiments the third and fourth sequence-specific recombinase target sites are selected from the group consisting of RS sites and Res sites, although other target sites are contemplated by the present invention. In some embodiments of the this method of the present invention, the first nucleic acid construct further comprises a third sequence-specific recombinase target site and the second nucleic acid constructs further comprises a fourth sequence-specific recombinase target site, wherein the method further comprises providing a second site-specific recombinase and the step of contacting the third nucleic acid construct with the second site-specific recombinase under conditions such that the third nucleic acid construct is recombined to form a fourth and a fifth nucleic acid construct.
The present invention also provides a recombined nucle
Elledge Stephen J.
Liu Qinghua
Baylor College of Medicine
Ketter James
Vinson & Elkins L.L.P.
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