Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-11-27
2003-05-27
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
Reexamination Certificate
active
06569678
ABSTRACT:
OTHER REFERENCES
Ahrenhotz et al., “A conditional suicide system in
Escherichia coli
based on intracellular degradation of DNA” Appl. Environ. Microbiol. 60, 3746-3751 (1994).
Altenbuchner et al., “Positive selection vectors based on palindromic DNA sequences” Methods Enzymol. 216, 457-466 (1992).
Bernard et al., “New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers” Gene 148, 71-74 (1994).
Bolivar et al., “Construction and characterization of new cloning vehicles, II. A multipurpose cloning system” Gene 2, 95-113 (1977).
Clark, J. M., “Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases” Nucl. Acids Res. 16, 9677-9686 (1988).
Henrich, B. and Plapp, R., “Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector” Gene 42, 345-349 (1986).
Henrich, B. and Schmidtberger, B., “Positive-selection vector with enhanced lytic potential based on a variant of phi X174 phage gene E” Gene 154, 51-54 (1995).
Holton, T. A. and Graham, M. W., “A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors” Nucl. Acids Res. 19, 1156 (1991).
Hu, G., “DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3′ end of a DNA fragment” DNA Cell Biol. 12, 763-770 (1993).
Kast, P., “pKSS—a second-generation general purpose cloning vector for efficient positive selection of recombinant clones” Gene 138, 109-114 (1994).
Kaufmann, D. L. and Evans, G. A., “restriction endonuclease cleavage at the termini of PCR products” BioTechniques 9, 304-306 (1990).
Kuhn et al., “Positive selection vectors utilizing lethality of EcoRI endonuclease” Gene 42, 252-263 (1986).
Malo, M. S. and Loughlin, R. E., “Promoter elements and regulation of expression of the cysD gene of
Escherichia coli
K-12” Gene 87, 127-131 (1990).
Mead et al., “Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template” BioTechniques 9, 657-663 (1991).
Messing et al., “Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro” Proc. Natl. Acad. Sci. 79, 3642-3646 (1977).
Mullis, K. B. and Faloona, F. A., “Specific synthesis of DNA in vitro via polymerase-catalyzed chain reaction” 1987, Methods Enzymol. 155, 335-350 (1987).
Norrander et al., “Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis” Gene 26, 101-106 (1983).
Pierce et al., “A positive selection vector for cloning high molecular DNA by bacteriophage P1 system: improved cloning efficiency” Proc. Natl. Acad. Sci. 89, 2056-2060 (1992).
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
Saiki et al., “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia” Science 230, 1350-1354 (1985).
Yanisch-Perron et al., “Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 pUC19 vectors” Gene 33, 103-119 (1985).
Yazynin et al., “A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene” Gene 169, 131-132 (1996).
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCHER DEVELOPMENT
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REFERENCE TO A MICROFICHE APPENDIX
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REFERENCE TO SEQUENCE LISTING
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FIELD OF THE INVENTION
The present invention relates to a positive selection vector system for direct cloning of PCR amplified nucleic acids. The invention involves insertional reconstruction of a reporter or of a regulatory gene. The invention describes reduction of exonuclease-induced false positive clones in a cloning experiment.
BACKGROUND OF THE INVENTION
Polymerase chain reaction or PCR (Saiki et al., 1985, Science 230, 1350-1354; Mullis and Faloona, 1987, Method Enzymol. 155, 335-350; U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188) is a milestone technological development in the field of molecular biology and genetic engineering. For amplification of a target nucleic acid PCR uses a polymerase, target sequence-specific forward and reverse primers, deoxynucleotides and a minute amount of target nucleic acid as the template. Repeated cycles of denaturation of double-stranded DNA followed by primer annealing and primer extension achieve an exponential amplification of the target DNA sequence.
The PCR product itself could be used for diagnosis, quantitation of the template, direct sequencing and some other applications (U.S. Pat. Nos. 5,856,144; 5,487,993 and 5,891,687). However, for applications such as mutation analysis, sequencing, gene expression, identification of polymorphic transcripts, making RNA probes etc., usually a large quantity of DNA is needed. Thus it is necessary to isolate a bacterial clone carrying the PCR generated target DNA fragment in a vector. Different methods for cloning PCR generated DNA fragments have been described. One such method involves incorporation of restriction endonuclease cleavage sites near the 5′ end of the PCR primers and the PCR product thus obtained is subjected to purification, restriction digestion with the respective endonuclease followed by ligation into a compatible vector, transformation and identification of the bacterial clone carrying the PCR fragment (Kaufmann and Evans, 1990, BioTechniques 9, 304-306).
The most common method for cloning a PCR product utilizes the nontemplate-dependent terminal transferase or extendase activity of Taq DNA polymerase, which usually produces a dAMP (deoxyadenosine monophosphate) overhang at the 3′ end of the PCR amplified DNA fragment (Clark, 1988, Nucl. Acid Res. 16, 9677-9686; Hu, 1993, DNA Cell Biol. 12, 763-770). The PCR product thus obtained is ligated into a linearized vector carrying a dTMP (deoxythymidine monophosphate) overhang at the 3′ end (U.S. Pat. No. 5,487,993; Mead et al., 1991, BioTechniques 9, 657-663; Holton and Graham, 1991, Nucl. Acids Res. 19, 1156). A similar strategy has been used when Taq polymerase generated PCR fragments carrying dAMP overhang at the 3′ end are ligated into a linearized vector carrying an inosine or uracil overhang at the 3′ end (U.S. Pat. No. 5,856,144).
The above-mentioned vectors lack the positive selection capability. Thus upon transformation, all host cells carrying either the recombinant vector (containing an insert) or the nonrecombinant vector (containing no insert DNA) grow in the desired medium at an equal growth rate. To differentiate between a host cell carrying only the nonrecombinant vector from the host cell carrying the recombinant vector, DNA fragment is usually inserted into a chromogenic gene, the product of which is inactivated thus rendering the recombinant colony white in a chromogenic medium. When the chromogenic gene is lacZ, the transformant carrying the nonrecombinant vector turns blue in the presence of X-gal, the substrate for the lacZ gene product &bgr;-galactosidase (Messing et al., 1977, Proc. Natl. Acad. Sci. 79, 3642-3646; Norrander et al., 1983, Gene 26, 101-106; Yanisch-Perron et al., 1985, Gene 33, 103-119). When the number of recombinant colonies are low and nonrecombinant colonies are high in a plate, then it becomes very difficult to differentiate the recombinant colonies from the non-recombinant colonies. High number of colonies also lead to contamination between the recombinant and nonrecombinant colonies. Insertion of a small DNA fragment sometimes can generate pale blue recombinant clones, which may not be differentiated from the pale blue nonrecombinant clones arising from nonuniform distribution of Xgal, especially when Xgal is spread on the surface of medium.
To ameliorate the problems associated with the chromogenic selection of the recombinant clones many vectors have been developed with positive selection capability allowing only the recombinant clones to grow in a selection medium. Most of these positive selection vectors have been developed based on insertional inac
Husain Zaheed
Malo Madhu Sudan
Loeb Bronwen M.
Ropes & Gray
SyntheGen Systems, Inc.
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