Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-11-13
2003-04-08
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024330
Reexamination Certificate
active
06544782
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
References Cited [Referenced By]
U.S. Pat. Documents
4683195
July, 1987
Mullis et al.
435/6
4683202
July, 1987
Mullis
435/91
4965188
Oct., 1990
Mullis et al.
435/6
5487993
Jan., 1996
Herrnstadt et al.
435/172
5856144
Jan., 1999
Mierendorf et al.
435/91
5891687
April, 1999
Schlieper et al.
935/172
5910438
June, 1999
Bernard et al.
435/252
OTHER REFERENCES
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Altenbuchner et al., “Positive selection vectors based on palindromic DNA sequences” Methods Enzymol. 216, 457-466 (1992).
Balbas et al., “Plasmid vector pBR322 and its special-purpose derivatives—a review” Gene 50, 3-40 (1986).
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).
Burns D. M. and Beacham, I. R., “Positive selection vectors: a small plasmid-vector useful for the direct selection of Sau3A-generated overlapping DNA fragments” Gene 27, 323-325 (1984).
Clark, J. M., “Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases” Nucl. Acids Res. 16, 9677-9686 (1988).
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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).
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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).
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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).
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FIELD OF THE INVENTION
The present invention relates to positive selection vectors for direct cloning of PCR-amplified nucleic acids. The invention also deals with modulation of regulatory elements for developing such vectors. The invention greatly reduces, if not eliminates, exonuclease-induced false positive clones in a DNA cloning experiment.
BACKGROUND OF THE INVENTION
Recent advances in the field of molecular biology and genetic engineering include polymerase chain reaction or PCR as described in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188. To amplify or reproduce copies of a targeted nucleic acid, PCR uses a polymerase, targeted sequence-specific forward and reverse primers, deoxynucleotides and a minute amount of target nucleic acid as the template. Exponential amplification of the targeted DNA sequence is achieved by repeated cycles of denaturation of double-stranded DNA followed by primer annealing and primer extension.
PCR-amplified DNA itself has been used for diagnosis, quantitation of the template DNA, direct sequencing and several other applications (U.S. Pat. Nos. 5,856,144; 5,487,993 and 5,891,687). However, for applications such as detection of polymorphism, mutations, sequencing, expression of genes, synthesis of RNA probes etc., it is often necessary to obtain a large quantity of DNA. This necessitates isolation of a bacterial clone carrying the PCR-generated targeted DNA fragment in a vector. Various strategies have been described for cloning PCR-generated DNA fragments into appropriate vectors. One such method involves incorporation of restriction endonuclease cleavage sites near the 5′ end of the PCR primers. 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 commonly used strategy involves 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). In a similar strategy, Taq DNA 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).
Since the above-mentioned vectors lack the positive selection capability, upon transformation, all host cells carrying either the recombinant vector (containing an insert) or the non-recombinant vector (containing no insert) grow in the desired medium at an equal growth rate. To differentiate between a host cell carrying the non-recombinant religated vector from the host cell carrying the recombinant vector, the DNA fragment to be cloned is usually inserted into a chromogenic gene, the product of which is thus inactivated rendering the recombinant colony white in a chromogenic medium. When the chromogenic gene is lacZ, the transformant carrying the non-recombinant 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
Husain Zaheed
Malo Madhu Sudan
Chunduru Suryaprabha
Fredman Jeffrey
Ropes & Gray
Synthegen Systems
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