Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-07-09
2002-03-12
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S091400, C435S320100, C435S252800, C435S252100, C435S325000, C536S023100
Reexamination Certificate
active
06355412
ABSTRACT:
TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. DESCRIPTION OF THE FIGS.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Methods for Cloning and Subcloning by Homologous Recombination
5.1.1 Approach 1: Introduction of Vector into Host Cell Containing Target DNA
5.1.2 Approach 2: Co-Introduction of Vector and Target DNA into the Host Cell
5.1.3 Approach 3: Introduction of Target DNA into Host Cells Containing Vector DNA
5.2 Compositions for Cloning and Subcloning by Homologous Recombination
5.2.1 The Homology Cloning Vector
5.2.1.1 The Origin of Replication
5.2.1.2 The Selectable Marker
5.2.1.3 the Homology Arms
5.2.1.4 Adapter Oligonucleotide Homology Arms
5.2.1.5 Construction of the Vector
5.2.2 Bacterial Recombinases
5.2.2.1 Protein Expression
5.2.3 Host Cells
5.2.4 Target DNA
5.3 Methods for use of the Invention
5.3.1 Introduction of DNA into Host Cells
5.3.2 Oligonucleotides
5.3.3 DNA Amplification
5.4 Methods for Diagnostic Applications
5.4.1 Detection of Foreign DNA
5.4.2 Diagnosis of Mutations and Polymorphisms in Cellular DNA
5.5 Kits
6. EXAMPLE: RECE/T AND RED&agr;/&bgr; SUBCLONING
6.1 Methods and Materials
6.2 Results
1. INTRODUCTION
The present invention is directed to methods and compositions for DNA cloning and subcloning using bacterial recombinase-mediated homologous recombination. In a specific embodiment, RecE/T or Red&agr;/&bgr; recombinases, or any functionally equivalent system for initiating bacterial homologous recombination, such as erf from phage P22, are used. In particular, the invention relates to cloning methods, diagnostic methods, compositions comprising polynucleotides useful as cloning vectors, cells comprising such polynucleotide compositions, and kits useful for RecE/T and Red&agr;/&bgr; mediated cloning.
2. BACKGROUND OF THE INVENTION
DNA cloning and subcloning in
E. coli
are fundamental to molecular biology. DNA cloning refers to the process whereby an origin of replication is operably linked to a double-stranded DNA fragment, and propagated in
E. coli,
or other suitable host. DNA subcloning refers to the process whereby a double-stranded DNA fragment is taken from a DNA molecule that has already been amplified, either in vitro, for example by PCR, or in vivo by propagation in
E. coli
or other suitable host, and is then linked to an operable origin of replication. Cloning and subcloning in
E. coli
is typically performed by ligating the ends of a DNA fragment to the ends of a linearized vector containing an
E. coli
origin of replication and a selectable marker. The selectable marker is included in the vector to ensure that the newly cloned product, the plasmid containing the insert, is retained and propagated when introduced into its
E. coli
host cell.
Conventional cloning methods have certain limitations. For example, since conventional cloning requires the use of restriction enzymes, the choice of DNA fragments is limited by the availability of restriction enzyme recognition sites in the DNA region of interest. Restriction sites must be found that cut the boundaries of, but not within, the desired DNA fragment. Since most useful restriction enzymes cut fairly frequently, the size of the linear DNA fragment made is also limited.
The increasing use of the polymerase chain reaction (PCR) for generating DNA fragments presents a second major drawback to conventional subcloning. The ends of PCR products are inefficient in ligation reactions due to non-templated nucleotides added to the 3′ termini of amplified PCR products by thermostabile polymerase. Furthermore, the use of PCR entails a high risk of mutations. Thus, molecular biologists have searched for new, more effective methods for cloning fragments of DNA, particularly when such fragments are longer than those conveniently accessible by restriction enzyme or PCR methodologies.
Homologous recombination is an alternative approach for cloning and subcloning DNA fragments. Methods for subcloning PCR products in
E. coli
that exploit the host's homologous recombination systems have been described (Oliner et al., 1993, Nucleic Acids Res. 21:5192-97; Bubeck et al., 1993, Nucl. Acids. Res. 21:3601-3602). In such methods, PCR primers, designed to contain terminal sequences homologous to sequences located at the ends of a linearized vector, are used to amplify a DNA fragment of interest. The PCR product and the linearized vector are then introduced into
E. coli.
Homologous recombination within the
E. coli
host cell results in insertion of the PCR product sequences into the plasmid vector. Although these methods have been shown to be useful for subcloning PCR fragments, they have not been applied to subcloning long DNA fragments, or to cloning DNA fragments of any size.
Another method describes an in vivo subcloning method in which two linear DNA molecules, one of which has an origin of replication, and which have long regions of homology at their ends, are used to transform an
E. coli
sbcBC host cell. Homologous recombination occurs in vivo, and results in circularization and propagation of the newly formed plasmid (Degryse, 1996, Gene 170:45). Subsequently, the ability of
E. coli
sbcBC host cells to mediate homologous recombination has been applied to subcloning large DNA fragments from adenovirus and herpes virus genomic DNAs (Chartier et al., 1996, J. Virol. 70: 4805; Messerle, et al., 1997, Proc. Natl. Acad. Sci. USA 94, 14759-14763; He, 1998, Proc. Natl Acad. Sci. USA 95:2509-2514). As described, each subcloning by homologous recombination in
E. coli
sbcBC host cells requires at least two preparatory subcloning steps to position long homology regions either side of an
E. coli
origin of replication. Furthermore, DNA cloning in
E. coli
sbcBC strains has not been described.
Recently, homologous recombination, mediated by either RecE/RecT (RecE/T) or Red&agr;/Red&bgr; (Red&agr;/&bgr;) has been shown to be useful for manipulating DNA molecules in
E. coli
(Zhang et al, 1998, Nature Genetics, 20, 123-128; Muyrers et al., 1999, Nucleic Acids Res. 27: 1555-1557). These papers show that, in
E. coli,
any intact, independently replicating, circular DNA molecule can be altered by RecE/T or Red&agr;/&bgr; mediated homologous recombination with a linear DNA fragment flanked by short regions of DNA sequence identical to regions present in the circular molecule. Integration of the linear DNA fragment into the circular molecule by homologous recombination replaces sequences between its flanking sequences and the corresponding sequences in the circular DNA molecule.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention provides methods and compositions for DNA cloning and subcloning using bacterial recombinase-mediated homologous recombination. The bacterial recombinase is preferably RecE/T and/or Red&agr;/&bgr;. Methods can be used to clone, subclone, propagate, and amplify a polynucleotide or mixture of polynucleotides of interest using a vector comprising short regions of DNA homologous to sequences flanking a designated target DNA sequence of interest and an origin of replication.
In one embodiment, the invention provides a method for introducing a double-stranded target DNA into a vector comprising culturing a bacterial cell that expresses a functional recombinase, said bacterial cell containing (a) the target DNA comprising a first double-stranded terminus and a second double-stranded terminus, and (b) a vector DNA comprising, in the following order along the vector DNA strand: (i) a first double-stranded homology arm (ii) an origin of replication; and (iii) a second double-stranded homology arm, such that the sequence of a vector DNA strand of the first homology arm is homologous to the sequence of a target DNA strand of the first terminus, and the sequence of a vector DNA strand of the second homology arm is homologous to the sequence of the target DNA strand of the second terminus, such that the target DNA
Muyrers Joep Pieter Paul
Stewart A. Francis
Zhang Youming
Davis Katharine F
Pennie & Edmonds LLP
Schwartzman Robert A.
The European Molecular Biology Laboratory
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