Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-08-30
2004-09-07
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252300, C435S320100
Reexamination Certificate
active
06787316
ABSTRACT:
DESCRIPTION
The invention refers to a novel method for cloning DNA molecules using a homologous recombination mechanism between at least two DNA molecules. Further, novel reagent kits suitable for DNA cloning are provided.
Current methods for cloning foreign DNA in bacterial cells usually comprise the steps of providing a suitable bacterial vector, cleaving said vector with a restriction enzyme and in vitro-inserting a foreign DNA fragment in said vector. The resulting recombinant vectors are then used to transform bacteria. Although such cloning methods have been used successfully for about 20 years they suffer from several drawbacks. These drawbacks are, in particular, that the in vitro steps required for inserting foreign DNA in a vector are often very complicated and time-consuming, if no suitable restriction sites are available on the foreign DNA or the vector.
Furthermore, current methods usually rely on the presence of suitable restriction enzyme cleavage sites in the vector into which the foreign DNA fragment is placed. This imposes two limitations on the final cloning product. First, the foreign DNA fragment can usually only be inserted into the vector at the position of such a restriction site or sites. Thus, the cloning product is limited by the disposition of suitable restriction sites and cloning into regions of the vector where there is no suitable restriction site, is difficult and often imprecise. Second, since restriction sites are typically 4 to 8 base pairs in length, they occur a multiple number of times as the size of the DNA molecules being used increases. This represents a practical limitation to the size of the DNA molecules that can be manipulated by most current cloning techniques. In particular, the larger sizes of DNA cloned into vectors such as cosmids, BACs, PACs and P1s are such that it is usually impractical to manipulate them directly by restriction enzyme based techniques. Therefore, there is a need for providing a new cloning method, from which the drawbacks of the prior art have at least partly been eliminated.
According to the present invention it was found that an efficient homologous recombination mechanism between two DNA molecules occurs at usable frequencies in a bacterial host cell which is capable of expressing the products of the recE and recT genes or functionally related genes such as the red&agr; and red&bgr; genes, or the phage P22 recombination system (Kolodner et al., Mol. Microbiol. 11 (1994) 23-30; Fenton, A. C. and Poteete, A. R., Virology 134 (1984) 148-160; Poteete, A. R. and Fenton, A. C., Virology 134 (1984) 161-167). This novel method of cloning DNA fragments is termed “ET cloning”.
The identification and characterization of the
E. coli
RecE and RecT proteins is described Gillen et al. (J. Bacteriol. 145 (1981), 521-532) and Hall et al. (J. Bacteriol. 175 (1993), 277-287). Hall and Kolodner (Proc. Natl. Acad. Sci. USA 91 (1994), 3205-3209) disclose in vitro homologous pairing and strand exchange of linear double-stranded DNA and homologous circular single-stranded DNA promoted by the RecT protein. Any references to the use of this method for the cloning of DNA molecules in cells cannot be found therein.
The recET pathway of genetic recombination in
E. coli
is known (Hall and Kolodner (1994), supra; Gillen et al. (1981), supra). This pathway requires the expression of two genes, recE and recT. The DNA sequence of these genes has been published (Hall et al., supra). The RecE protein is similar to bacteriophage proteins, such as &lgr; exo or &lgr; Red&agr; (Gillen et al., J. Mol. Biol. 113 (1977), 27-41; Little, J. Biol. Chem. 242 (1967), 679-686; Radding and Carter, J. Biol. Chem. 246 (1971), 2513-2518; Joseph and Kolodner, J. Biol. Chem. 258 (1983), 10418-10424). The RecT protein is similar to bacteriophage proteins, such as &lgr; &bgr;-protein or &lgr; Red&bgr; (Hall et al. (1993), supra; Muniyappa and Radding, J. Biol. Chem. 261 (1986), 7472-7478; Kmiec and Hollomon, J. Biol. Chem. 256 (1981), 12636-12639). The content of the above-cited documents is incorporated herein by reference.
Oliner et al. (Nucl. Acids Res. 21 (1993), 5192-5197) describe in vivo cloning of PCR products in
E. coli
by intermolecular homologous recombination between a linear PCR product and a linearized plasmid vector. Other previous attempts to develop new cloning methods based on homologous recombination in prokaryotes, too, relied on the use of restriction enzymes to linearise the vector (Bubeck et al., Nucleic Acids Res. 21 (1993), 3601-3602; Oliner et al., Nucleic Acids Res. 21 (1993), 5192-5197; Degryse, Gene 170 (1996), 45-50) or on the host-specific recA-dependent recombination system (Hamilton et al., J. Bacteriol. 171 (1989), 4617-4622; Yang et al., Nature Biotech. 15 (1997), 859-865; Dabert and Smith, Genetics 145 (1997), 877-889). These methods are of very limited applicability and are hardly used in practice.
The novel method of cloning DNA according to the present invention does not require in vitro treatments with restriction enzymes or DNA ligases and is therefore fundamentally distinct from the standard methodologies of DNA cloning. The method relies on a pathway of homologous recombination in
E. coli
involving the recE and recT gene products, or the red&agr; and red&bgr; gene products, or functionally equivalent gene products. The method covalently combines one preferably linear and preferably extrachromosomal DNA fragment, the DNA fragment to be cloned, with one second preferably circular DNA vector molecule, either an episome or the endogenous host chromosome or chromosomes. It is therefore distinct from previous descriptions of cloning in
E. coli
by homologous recombination which either rely on the use of two linear DNA fragments or different recombination pathways.
The present invention provides a flexible way to use homologous recombination to engineer large DNA molecules including an intact>76 kb plasmid and the
E. coli
chromosome. Thus, there is practically no limitation of target choice either according to size or site. Therefore, any recipient DNA in a host cell, from high copy plasmid to the genome, is amenable to precise alteration. In addition to engineering large DNA molecules, the invention outlines new, restriction enzyme-independent approaches to DNA design. For example, deletions between any two chosen base pairs in a target episome can be made by choice of oligonucleotide homology arms. Similarly, chosen DNA sequences can be inserted at a chosen base pair to create, for example, altered protein reading frames. Concerted combinations of insertions and deletions, as well as point mutations, are also possible. The application of these strategies is particularly relevant to complex or difficult DNA constructions, for example, those intended for homologous recombinations in eukaryotic cells, e.g. mouse embryonic stem cells. Further, the present invention provides a simple way to position site specific recombination target sites exactly where desired. This will simplify applications of site specific recombination in other living systems, such as plants and mice.
A subject matter of the present invention is a method for cloning DNA molecules in cells comprising the steps:
a) providing a host cell capable of performing homologous recombination,
b) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and
c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.
In the method of the present invention the homologous recombination preferably occurs via the recET mechanism, i.e. the homologous recombination is mediated by the gene products of the recE and the recT genes which are preferably selected from the
E. coli
genes recE and recT or functionally related genes such as the phage &
Buchholz Frank
Stewart Francis
Zhang Youming
Europaisches Laboratorium fur Molekularbiologie (EMBL)
Fredman Jeffrey
Rothwell Figg Ernst & Manbeck P.C.
Strzelecka Teresa
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