Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-02-26
2004-02-24
Leffers, Jr., Gerald G. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S029000, C435S252300, C435S252330, C435S320100, C435S325000, C435S091100, C435S091410, C435S091420, C435S471000, C435S472000, C435S440000, C536S023100
Reexamination Certificate
active
06696278
ABSTRACT:
BACKGROUND OF THE INVENTION
The most common manipulation of vectors in molecular biology laboratories is the transfer of a gene of interest into a vector of choice. The resulting recombinant vectors allow specialized applications such as expression in mammalian cells, expression in bacterial hosts, purification of the native protein through employment of specialized (vector provided) purification tags or detection of interaction with other proteins (two-hybrid systems). Typically, cloning is achieved through restriction digestion, isolation of the desired fragments and reconstitution of the desired plasmid by ligation. Although this technique has been routinely employed for approximately 20 years, it is still error-prone and cumbersome.
There is a need in the art for a method of transferring a desired coding region to a vector of interest without the use of restriction enzyme recognition sites and restriction enzymes. In prior art methods, multiple restriction enzymes are 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. More recently, recombinase-based cloning methods have been developed. However, the current methods require multiple recombination events.
There is a need in the art for cloning of newly discovered sequences, such as new genes. Thus there is a need in the art for more efficient techniques for transfer of the genes of interest into a vector of choice. It is desirable that such a technique permits high fidelity, high efficiency and a minimum number of handling steps to allow adaptation to automated procedures.
There is a need in the art for a method for the cloning of a DNA molecule which permits rapid transfer of the DNA molecules from one vector to another without the need to rely upon restriction enzyme digestions.
SUMMARY OF THE INVENTION
The present invention provides a method of transfer of a gene of interest to a product vector comprising: contacting in vitro (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon, and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (3) a site-specific recombinase, wherein the contacting permits formation of a co-integrate vector comprising the first and the second vector. The co-integrate vector is subsequently introduced into a prokaryotic host cell so as to permit the formation of a product vector comprising the gene of interest interposed between the double-stranded origin of replication of the second vector and the site-specific recombination recognition site, the single-stranded origin of replication of the second vector, and the gene encoding the second selectable marker, wherein the product vector does not include both of the gene encoding the negative selectable marker and the gene encoding the first selectable marker.
The present invention further provides a method of transfer of a gene of interest to a co-integrate vector comprising contacting in vitro (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon; and (c) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (3) a site-specific recombinase, wherein the contacting permits formation of a co-integrate vector comprising the first and the second vector.
In one embodiment, the co-integrate vector is introduced into a prokaryotic host cell.
The present invention further provides a method of transfer of a gene of interest to a product vector comprising introducing into a prokaryotic host cell which expresses a gene encoding a site-specific recombinase (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon; and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site, and wherein said prokaryotic host cell further expresses a gene encoding a rep protein which can initiate replication at the double stranded origin of replication. The introduction of the first and second vector to the prokaryotic host cell permits formation of a product vector comprising the gene of interest interposed between the double-stranded origin of replication of the second vector and the site-specific recombination recognition site, the single-stranded origin of replication of the second vector, and the gene encoding the second selectable marker, the product vector not including both of the negative selectable marker and the gene encoding the first selectable marker.
The present invention further provides a method of transfer of a gene of interest to a co-integrate vector comprising introducing into a prokaryotic host cell which expresses a gene encoding a site-specific recombinase a first vector and a second vector so as to permit recombination of the first and second vectors to produce a co-integrate vector, wherein the first vector comprises (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon, and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and the second vector comprises (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of repli
Leffers, Jr. Gerald G.
Palmer & Dodge LLP
Stratagene
Williams Kathleen M.
LandOfFree
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