WO2002042446A1 - Prgr: a positive selection vector system for direct cloning of pcr amplified dna fragments - Google Patents

Abstract

The present invention describes a positive selection vector based on insertional reconstruction of a reporter gene or of a regulatory gene controlling the expression of a reporter gene. The cloning vector carries a reporter gene or a regulatory gene with a dominant mutation at its 3' or 5' end thus rendering the reporter or the regulatory gene protein functionally inactive. A primer carrying a nucleic acid sequence that corrects the mutation is used during PCR amplification of a targeted nucleic acid sequence, and the amplified DNA fragment is then ligated to the cloning vector thus reconstructing the wild-type reporter or regulatory gene. Two positive selection vectors pRGR1Ap and pRGR2Ap have been constructed for use in a positive selection cloning method. Use of pRGR1Ap and pRGR2Ap greatly reduce exonuclease-induced false positive clones.

Description

pRGR: A POSITIVE SELECTION VECTOR SYSTEM FOR DIRECT CLONING OF PCR AMPLIFIED DNA FRAGMENTS

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. Patent 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. Patent 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 (Kauftnann 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. Patent No. 5,487,993; Mead et al, 1991, BioTechniques 9, 657-663; Holton and Graham, 199 1, 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. Patent No. 5,856,144).

The above-mentioned vectors lack the positive selection capability. Thus upon transformation, all host cells carrying either the recombmant 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 β-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 inactivation of lethal genes (Pierce et al, 1992, Proc. Natl. Acad. Sci. 89, 2056-2060; Henrich and Plapp, 1986, Gene 42, 345-349; Henrich and Schmidtberger, 1995, Gene 154, 51-54; Bernard et al., 1994, Gene 148, 71-74; Kuhn et al., 1986, Gene 42, 253-263; U.S. Patent No. 5,910,438; U.S. Patent No. 5,891,687). A vector system based on abolition of sensitivity towards metabolite has also been described (Kast, 1994, Gene 138,109-114). Vectors have also been constructed based on selection by means of DNA-degrading or RNA-degrading enzymes (Yazynin et al., 1996, Gene 169, 131-132; Ahrenhotz et al., 1994, Appl. Environ. Microbiol. 60, 3746-3751) as well as based on selection by destruction of long palindromic DNA sequences (Altenbuchner et al., 1992, Methods Enzymol. 216, 457-466).

The presently available positive selection vectors as well as other cloning vectors are associated with many disadvantages. An inherent problem of a vector with a lethal or a chromogenic gene is a high number of false positive clones, i.e., clones without any insert. The false positive clones could be revertants arising out of dominant mutations in the lethal or chromogenic gene rendering it inactive. However, the biggest disadvantage of every cloning system available today is the exonuclease-induced generation of false positive clones. The reagents used in restriction digestion, PCR and ligation, such as restriction enzymes, polymerases and ligases, are usually contaminated with exonucleases, which are seldom completely removed from larger lots of commercial preparations. Exonuclease digestion deletes some nucleotide bases from the cloning site in the chromogenic or lethal gene in a linearized vector DNA. Thus recircularization of such vectors results in inactivation of the chromogenic or lethal genes, and upon transformation, these recircularized vectors give false positive transformant clones. A palindromic sequence could also be destroyed by exonuclease digestion resulting in generation of false positive clones.

Insertion of a small DNA fragment in frame with the nucleotide sequence of the lethal gene or the chromogenic gene may in some cases not alter the function of the lethal or chromogenic gene, thus making it impossible to clone such small DNA fragments. Furthermore, when cloning of a small DNA fragment results in diminished function of the lethal gene, which nevertheless remains functional, then the recombinant clones grow at a reduced rate in case of positive selection vectors, and these clones could be confused with the non-recombinant clones growing because of diminished selection pressure due to, for example, long period of incubation. A further disadvantage of the vectors based on lethal genes is that it may require a complex medium to activate the selection mechanism (Kast, 1994, Gene 138, 109-114). The positive selection vectors carrying lethal or chromogenic genes also require special host cells for transformation, e.g., CcdB based vectors require F^~~ host cells (U.S. Patent No. 5,910,438), CAP based vectors require adenyl-cyclase positive host cells (U.S. Patent No. 5,891,687) and lacZ based vectors require lac^~ host cells (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). A special regulatory system, usually lad or CI repressor system (U.S. Patent No. 5,910,438; Pierce et al., 1992, Proc. Natl. Acad. Sci. 89, 2056- 2060), has also to be in place to prevent the expression of the lethal gene in the host cell during the preparation of vector DNA.

OBJECTS OF THE INVENTION

The primary object of the present invention is to develop a simple cloning and/or sequencing vector having the capability of positive selection thus allowing only the recombinant clones (carrying an insert DNA) to grow in a selection medium, whereas, the non-recombinant clones (carrying no insert DNA) would not grow. The vector could also be used as a positive selection expression vector.

The particular object of the present invention is to eliminate or greatly reduce the generation of false positive clones associated with all the presently available cloning systems. Especial emphasis is given to the elimination of exonuclease-induced false positive clones. Thus the present invention aims to apply the principle of insertional reconstruction of a reporter gene or a regulatory gene controlling the expression of a reporter gene. It was aimed to develop a positive selection vector based on insertional reconstruction of an antibiotic resistance reporter gene, which carries a dominant negative mutation at its 5' or 3' end. Thus upon transformation non-recombinant clones will not grow in presence of the respective antibiotic. Insertional reconstruction allows correction of the mutation in the antibiotic resistance reporter gene. Thus upon transformation of a host cell the reconstructed reporter gene produces functionally active antibiotic resistance reporter gene protein thus allowing the host cell to grow in a specific selection medium containing the respective antibiotic.

Use of the principle of reconstruction of a reporter gene should also greatly reduce, if not eliminate, revertants because firstly, probability of spontaneous mutational reconstruction of the wild-type reporter or regulatory gene is minimal, and secondly, any mutation in the coding sequence of the reporter or regulatory gene would rather negatively affect the function of the respective gene protein.

A vector system based on antibiotic resistance gene as the reporter gene should also eliminate the need of any special type of host cells. The elimination of the disadvantages associated with the presently available vectors is greatly desirable. A vector system based on reporter gene reconstruction will mostly eliminate these disadvantages and hence will be a substantial technological achievement.

SUMMARY OF THE INVENTION The present invention relates to a strategy for developing positive selection vectors based on reconstruction of a reporter gene or of a regulatory gene controlling the expression of a reporter gene. The invention also describes the use of such vectors for direct cloning of PCR products. As an example of application of the strategy, a positive selection vector pRGRl Ap has been developed. When the last (position 286) amino acid tryptophan (encoded by 5 '-TGG-3) of ampicillin resistance gene β-lactamase is replaced by valine (encoded by 5-GTG-3') β-lactamase becomes functionally inactive. The sequence 5'- GTG-3' is a part of the Pml I restriction endonuclease cleavage site 5'-CACGTG-3', which is a unique cloning site in this vector. Thus upon Pml I restriction endonuclease cleavage 5'-CAC-3' and 5'-GTG-3' are created at the 3' and 5' ends respectively of the linearized vector. A PCR primer carrying the nucleotides 5 '-TGGTAA-3 ' at its 5 ' end is used in

PCR. When the resulting blunt-ended PCR products thus obtained are ligated to the vector the reporter ampicillin resistance gene is reconstructed correcting the mutation. The nucleotides 5'-TAA-3' constitute the stop codon for the β-lactamase gene. Subsequent transformation of a host cell with the recombinant vector (carrying an insert DNA) produces functionally active β-lactamase, which confers resistance to ampicillin.

Restriction sites of Cla I, EcoR V, Nar I, Nde I, Not I and Sfi I have been introduced in this vector for easy extraction of the insert. The Nar I site in the β-lactamase gene does not change the amino acid sequence of β-lactamase. Introduction of EcoR V restriction site in β-lactarnase gene (bla) changes glutarnic acid at position 277 into aspartic acid, whereas, the restriction site Nde I changes isoleucine into methionine at position 275, and glutamine into histidine at position 274. The restriction site Cla I in β-lactamase gene (bla) replaces methionine at position 268 with isoleucine. The mutations introduced by restriction sites Nar I, EcoR V, Nde I and Cla I do not have any significant effect on the function of β- actamase. Another vector pRGR2Ap, which is similar to pRGRlAp, has been described. The vector pRGR2Ap contains pUC origin of replication, M13 origin of replication and T7 phage promoter.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an exemplary positive selection vector pRGRlAp, constructed according to the principle of reporter gene reconstruction. The ampicillin resistance gene is inactive due to a dominant negative mutation at its 3' end. FIG. 2 shows the nucleotide sequence around the cloning site Pml I in pRGRlAp.

An asterisk indicates a position of mutation in the ampicillin resistance gene.

FIG. 3 shows the restriction map of pBR322Δropl.

FIG. 4 shows the restriction map of pBR322ΔropIM13.2.

FIG. 5 shows the restriction map of pRGR2Ap. The ampicillin resistance gene is inactive due to a dominant negative mutation at its 3' end.

FIG. 6 shows the nucleotide sequence around the cloning site Pml I in pRGR2Ap.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to construct a positive selection vector system based on reconstruction of a reporter gene or a regulatory gene controlling the expression of a reporter gene. The invention also describes the use of such a vector for direct cloning of PCR generated DNA fragments. The vector carries a viral or plasmid origin for autonomous replication in an appropriate host cell. The vector contains an M 13 or fl origin of replication for easy isolation of single-stranded form of plasmid DNA upon coinfection of the host cells with a helper phage. It also carries a selectable marker gene, a reporter and/or regulatory gene with a dominant negative mutation, and a cloning site for inserting a PCR amplified DNA fragment or a restriction DNA fragment.

The invention envisions development of chromogenic or fluorogenic selection vectors based on reconstruction of the of lacZ or fluorescent protein genes. The invention also contemplates insertional reconstruction of a regulatory gene controlling the expression of lacZ or a fluorescent protein gene. The selectable marker gene, which allows contamination-free growth of the host cells harboring the vector, is usually an antibiotic resistance gene, however, it could be an essential gene for the host or the vector itself A chromogenic gene, such as lacZ or a fluorogenic gene, such as GFP (Green Fluorescent Protein) gene can also serve as a selectable marker gene.

The vector carries a reporter gene, the function of which could easily be assayed either qualitatively or quantitatively. Like the selectable marker gene, the reporter gene could be an antibiotic resistance gene, a toxic gene, an essential gene for the host or the vector, and a chromogenic gene, such as lacZ or a fluorogenic gene, such as a fluorescent protein gene.

When a positive selection vector is constructed based on insertional reconstruction of a reporter gene, the reporter gene is mutated in this vector so that upon transformation of a host cell the vector is unable to produce any functionally active reporter gene protein thus rendering the host cell unable to grow in a specific medium. When the positive selection vector is constructed based on insertional reconstruction of a regulatory gene, the reporter gene in this vector is functional, whereas, the regulatory gene carries the dominant negative mutation. Insertional reconstruction of the regulatory gene allows the host cell carrying the recombinant plasmid (containing an insert) to grow in a specific medium.

When the reporter gene is an antibiotic resistance gene and the vector is developed based on reconstruction of the reporter gene, the vector cannot produce the antibiotic resistance reporter gene protein resulting in inhibition of the growth of host cell in a' specific selection medium containing the respective antibiotic. Only insertional reconstruction of the reporter gene will ensure production of the antibiotic resistance reporter gene protein thus allowing a host cell harboring the recombinant vector (carrying an insert DNA) to grow in a selection media containing the respective antibiotic.

A positive selection vector can be developed based on insertional reconstruction of a regulatory gene controlling the expression of a reporter gene. A vector can carry a reporter gene under the control of a positively regulated promoter, for example the cysD promoter (Malo and Loughlin, 1990, Gene 87, 127-13 1), or under the control of a negatively regulated promoter, for example, the lac promoter. The cysD promoter in E. coli is positively regulated by the positive regulatory (activator) CysB protein, which means binding of CysB protein to the cysD promoter initiates transcription from the cysD promoter. The lac promoter for lacZYA operon in E. coli is negatively controlled by the negative regulatory (repressor) Lad protein, which means binding of the Lad protein to the lac operator stops transcription from the lac promoter. Different combinations of regulatory genes and reporter genes could be used to develop multiple positive selection vectors. A positive selection vector could be developed carrying an antibiotic resistance reporter gene under the control of cysD promoter, and the cysB gene as the regulatory gene, wherein the cysB gene carries a dominant negative mutation. Only insertional reconstruction of the cysB gene will allow production of the antibiotic resistance reporter gene protein resulting in growth of only a host cell harboring a recombinant (carrying an insert DNA) clone in presence of the respective antibiotic. Similarly, a positive selection vector could be developed carrying a toxic reporter gene, e.g., ccdB gene, under the control of lac promoter, and the lad gene as the regulatory gene, wherein the lad gene carries a dominant negative mutation. Only insertional reconstruction of the lad gene will inhibit production of the toxic gene protein resulting in growth of only a host cell harboring a recombinant (carrying an insert DNA) clone in a specific medium.

Chromogenic or fluorogenic selection vectors can be developed based on reconstruction of the reporter gene lacZ or fluorescent protein genes (e.g., GFP). LacZ or fluorescent protein genes under the control of a positive or a negative regulatory gene could also be used to develop chromogenic or fluorogenic selection vectors. Positive selection vectors pRGRl Ap and pRGR2Ap have been developed based on insertional reconstruction of an antibiotic resistance reporter gene. These vectors carry a dominant mutation at the 3' end of the ampicillin resistance gene (bla) and the vectors carry an unique cloning site Pml I. When a PCR fragment carrying 5'-TGGTAA-3' at its 5' end is inserted into the Pml I digested vector then the ampicillin resistance gene is reconstructed. Subsequent transformation of a host cell with a recombinant vector results in production of ampicillin resistance gene protein β-lactamase and thus the transformant grows in presence of ampicillin. The vectors have been successfully used to clone PCR fragments amplified by Pfu or Taq DNA polymerase. EXAMPLE 1

General Techniques of Molecular Biology

Unless otherwise indicated, the molecular biology techniques related to this invention are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

For enzymatic amplification of a targeted DNA fragment PCR was performed (Saiki et al, 1985, Science 230, 1350-1354; Mullis and Faloona, 1987, Method Enzymol. 155, 335-350) using a DNA thermal cycler (Perkin Elmer Cetus, Foster City, CA, USA) according to the manufacturers instructions. The thermostable DNA polymerases and the PCR kit (Perkin Elmer Cetus, Foster City, CA, USA; Stratagene, La Jolla, CA, USA) were used according to the recommendations of the respective suppliers.

DNA restriction digestion was performed according o the specifications of the manufacturers (New England Biolabs, Beverly, MA, USA; Stratagene, La Jolla, CA, USA). After restriction digestion the restriction endonuclease was inactivated by heat treatment or by phenol: chloroform: isoamyl alcohol (25:24: 1) treatment followed by ethanol precipitation of DNA. The precipitated DNA was then pelleted by centrifugation and the pellet was dried in air.

DNA ligation was achieved by incubating the DNA (vector and/or insert) in presence of T4 DNA ligase according to the instructions of the manufacturer (Life Technologies/GIBCO-BRL, Rockville, MD, USA).

Transformations of the commercially available competent host cells were carried out as per the instructions of the supplier (Life Technologies/GIBCO-BRL, Rockville, MD, USA). The transformants were plated onto LB agar medium (Life Technologies/GIBCO- BRL, Rockville, MD, USA) containing ampicillin (100 [tg/ml, Sigma, St. Louis, MO, USA) and/or tetracycline (12.5 μg/ml, Sigma, St. Louis, MO, USA). For small or large scale preparation of transformant plasmid DNA, cells were grown on LB broth (Life Technologies/GIBCO-BRL, Rockville, MD, USA) containing ampicillin (100 μg/ml) and/or tetracycline (12.5 μg/ml) and DNA was prepared using alkaline lysis method. The plasmids pUC 19 (Norrander et al., 1983, Gene 26, 101-106) and pBR322

(Bolivar et al., 1977, Gene 2, 95-113) DNA were purchased from New England Biolabs (Beverly, MA, USA) and E. coli DNA was obtained from Sigma (St. Louis, MO, USA). Restriction endonucleases were purchased from New England Biolabs (Beverly, MA, USA) and Stratagene (La Jolla, CA, USA). Taq DNA polymerase and dNTPs were purchased from Perkin Elmer Cetus (Foster City, CA, USA), and T4 DNA ligase was obtained from Life Technologies/GIBCO-BRL (Rockville, MD, USA). Pfu and Taqplus DNA polymerases were obtained from Stratagene (La Jolla, CA, USA). Oligonucleotides were synthesized by Biosource International (Camarillo, CA, USA). Kit for plasmid DNA extraction from agarose gel was purchased from Qiagen (Valencia, CA, USA). Construction of the Positive Selection Vector pRGRlAp

It was aimed to generate a dominant negative mutant of the ampicillin resistance gene β-lactamase (bla) as well as to create restriction endonuclease cleavage sites in its 3' coding region. It was decided to introduce the restriction sites in β-lactamase by PCR- mediated mutagenesis, followed by testing the effect of each mutation.

The following forward PCR primer RGR1F was synthesized to introduce Pml I, Nar I, EcoR V, Nde I and Cla I sites in the 3' coding region of β-lactamase. Introduction of the unique Pml I restriction site (5'-CACGTG-3') replaces the last amino acid (tryptophan at position 286, encoded by 5'TGG-3') of β-lactamase with a valine (encoded by 5'-GTG-3'). The Nar I site in the β-lactamase gene does not change the amino acid sequence of β- lactamase. Introduction of EcoR V restriction site in β-lactamase gene changes glutamic acid in position 277 into aspartic acid, whereas, the restriction site Nde I changes isoleucine into methionine in position 275, and glutamine into histidine in position 274. The restriction site Cla I in β-lactamase gene (bla) replaces methionine in position 268 with isoleucine.

The following reverse PCR primer RGRTR was synthesized to introduce restriction sites of Cla 1, EcoR V, Nar I, Nde I, Not I and Sfi I for easy extraction of the insert. Repeats of stop codons in all three reading frames were also introduced downstream of the β-lactamase gene. These stop codons would ensure that translation will be prematurely terminated in a recircularized non-recombinant vector, which would have been subjected to some exonuclease digestion. Thus the reporter gene protein will remain functionally inactive in a recircularized non-recombinant vector and minimize exonuclease-induced false positive clones. Forward primer RGRIF :

5'-CAATTACAC GTG CTTAAT CAGTGA GGC GCC GATATC AGC CAT ATGTCTATT TCGTTC ATC GATAGT TGC CTG-3' (96 bases) Reverse primer RGRIR:

5'- ATT AAG CAC GTG TAA TTG AAT AAT AGT TGA ATA GTA ATT GAA TAA GGC GCC ATA TGA TAT CGA TGG CCT AAG CGG CCG CTG TCA GAC CAA GTT TAC TCA TAT ATA CTT TAG-3' (111 bases) The PCR conditions were: l ng pBR322 DNA

10 μM forward primer RGRIF

10 μM reverse primer RGRIR

0.2 mM dNTPs (equimolar mixture of dATP, dGTP, dCTP and dTTP) 2.5 μl of 1 OX low salt buffer for Taqplus DNA polymerase.

2.5 U Taqplus DNA polymerase (Stratagene, La Jolla, CA, USA)

Distilled water making total volume up to 25 μl.

Two drops of mineral oil was added to overlay on the PCR mixture to prevent evaporation during PCR cycling. The PCR cycle conditions were as follows:

2 min at 94°C, then 15 cycles with: 1 min at 94°C, 1 min at 55°C, 8 min at 72°C; followed by a final extension step of 10 min at 72°C.

To verify the PCR reaction, 5 μl of the PCR product was electrophoresed in 0.8% agarose (Life Technologies/GIBCO-BRL, Rockville, MD, USA) gel in presence of ethidium bromide (Sigma, St. Louis, MO, USA) for about 1 hr, and the gel was then photographed under U-V light using Polaroid Type 667 films (Fisher Scientific, Suwanee, GA, USA). The rest of the PCR amplified DNA was treated with phenol:chloroform:isoamyl alcohol (25:24: 1) and was then precipitated by ethanol. Precipitated DNA was pelleted by centrifugation and the pellet was dried in air. The dried DNA was dissolved in 25 μl of IX Pml I restriction buffer and then incubated in presence of 20 U of Pml I restriction endonuclease for I hr at 370C. The digested DNA was electrophoresed in 0. 8% agarose gel. The desired DNA band was excised and DNA was purified using Qiaex kit. The purified DNA was used in ligation as per conditions given below: 15 μl of purified DNA

4 μl of 5X ligation buffer

1 μl (5 U) of T4 DNA ligase (Life Technologies/GIBCO-BRL, Rockville, W, USA) Ligation was performed for overnight at 160C.

An aliquot of 2 μl ligation mix was used to transform 50 μl Maxefficiency DH5a (Life Technologies/ GIBCO-BRL, Rockville, MD, USA) E. coli host cells according to the recommended protocols. The transfoπnants, were then plated onto LB agar plates containing 12.5 μl/ml tetracycline and incubated at 37°C overnight. Some transformant colonies were individually transferred to LB agar plates containing 100 μg/ml ampicillin as well as to LB agar plates containing 12.5 μg/ml tetracycline. The clones sensitive to ampicillin were then individually grown in 5 ml aliquot of LB broth containing 12.5 μg/ml tetracycline. Small scale plasmid DNA was isolated from each individual clone, and DNA was then digested with 20 U Pml I restriction endonuclease. Any plasmid DNA carrying a Pml I cleavage site was fm-fher characterized for the presence of other expected restriction endomiclease cleavage sites. One such plasmid carrying expected restriction endonuclease cleavage sites was named as pRGRlAp and the restriction map of this vector is shown in FIG. 1. The vector pRGRlAp is sensitive to ampicillin, and hence carries a dominant negative mutation in the 3' coding region of β-lactamase because of mutations introduced by the restriction sites. FIG. 2 shows the DNA sequence indicating the positions of mutations in β-lactamase gene in PRGRlAp, and also the mutated and other related restriction sites.

To test the effect of ^Trp286v_aι mutation (created by Pml I) it was decided to clone a PCR fragment carrying 5'-TGGTAA-3' at its 5' end into the Pml I digested pRGRlAp resulting in correction of the mutation. Large scale DNA of pRGRlAp was prepared using plasmid kit from Qiagen. An aliquot of 2 μg of pRGRlAp DNA was digested with 20 U of Pml I restriction endonuclease for 1 hr at 37°C. The digest was then incubated at 70°C for 30 min to inactivate Pml I, and was diluted with sterile DEPC-treated water to give final concentration 10 ng/μl. The vector pRGRlAp thus prepared was tested for direct cloning of PCR products as well as for its capability as a positive selection vector.

Example of Direct Cloning of PCR Product into pRGRlAp

A 420 bp fragment of the lacZ was separately PCR-amplified using Taq DNA polymerase (without 3'-5' proofreading exonuclease activity) and Pfu DNA polymerase (with 3'-5' proof reading exonuclease activity). Following are the primers used in amplification of the above mentioned 420 bp DNA fragment:

Forward primer LC1261RGRF:

5'-TGGTAA GCT TGC GGC CGC AAAGGC CAC AAT TTC ACA CAGGAA ACA GCTATG-3' (51 bases)

Reverse primer LC168OR:

5'-TTT CAT CAA CAT TAA ATG TGA GCG AGT AAC-3' (30 bases)

The forward primer LC 126 IRGRF carries 5-TGGTAA-3'at its 5' end and Not I and Sfi I sites for elucidation of the orientation of the insert in a recombinant plasmid. The PCR conditions were:

1 μg E.coli DNA

10 μM forward primer (LCI 26 IRGRF)

10 μM reverse primer (LC1680R)

0.2 mM dNTPs (equimolar mixture of dATP, dGTP, dCTP and dTfP) 2.5 μl of 1 OX buffer for Taq or Pfu DNA polymerase

2.5 U Taq or Pfu DNA polymerase

Distilled water making total volume up to 25 μl.

Two drops of mineral oil was added to overlay on the PCR mixture to prevent evaporation during PCR cycling. The PCR cycle conditions were as follows:

2 min at 94°C, then 25 cycles with: 1 min at 94°C, I min at 55°C, 2 min at 72°C; followed by a final extension step of 5 min at 72°C.

To verify the PCR reaction, 5 μl of the PCR product was electrophoresed in 1.5% agarose gel in presence of ethidium bromide for about 1 hr, and the gel was then photographed under UV light using Polaroid Type 667 films.

Ligation was performed using 1-200 ng of Pml I digested pRGRlAp vector DNA, and 1-5 μl of the PCR product. The conditions of atypical ligation experiment is given below:

1 μl (50 ng) of Pml I digested vector pRGRlAp DNA 2 μl (200 ng) of PCR product

4 μl of 5X ligase buffer 12 μl of DEPC-treated water l μl (5 U) ofT4 DNA ligase

Ligation mixture was incubated at room temperature for 5-30 min or at 16°C for overnight.

Different amounts (2- 10 μl) of ligation mix were used to transform 10- 100 μl of competent Maxefficiency DH5α E. coli cells (Life Technologies/GIBCO-BRL, Rockville, MD, USA). In a typical transformation 50 μl of DH5α was transformed with 2 μl of ligation mixture for 30 min on ice, followed by heat shock at 42°C for 50 sec, 2 min on ice, addition of 1.0 ml of SOC medium (Life Technologies/GIBCO-BRL, Rockville, MD, USA), and incubation at 37°C for 1 hr. An aliquot of 25-500 μl of the transformation mix was plated onto LB agar plates containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline and incubated overnight at 37°C. As expected, control ligation sample of vector alone gave only a few transformant, whereas, ligation samples of vector plus PCR fragments gave many transformant colonies. The result of this typical cloning experiment is shown in Table 1. Some transformant clones were individually cultured in 5 ml LB broth containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline for overnight at 37°C. Small scale plasmid DNA was isolated using standard alkaline lysis method (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The isolated plasmid DNA was characterized by Not I or Sfi I restriction endonuclease digestion, which should release the insert fragment.

TABLE 1

The positive selection vector pRGRl Ap was digested with Pml I restriction enzyme, and after heat inactivation of Pml I the vector DNA was used in ligation. Transformants were grown in presence of 100 μg/ml ampicillin and 12.5 μg/ml tetracycline. c The plasmids from the transformants were digested with either Not I or Sfi I releasing the insert. As expected, Table I shows that the vector pRGRlAp is capable of cloning the PCR fragments carrying 5'-TGGTAA-3' at its 5' end as produced by primers LC1261RGRF and LC1680R. Table 1 also shows that the vector pRGRlAp is capable of cloning both types of PCR products produced by Taq and Pfu DNA polymerases. However, fewer number of clones are obtained in case of cloning of the PCR products obtained by Taq DNA polymerase because in such case only those PCR products of Taq DNA polymerase are ligated which have no dAMP overhang. Improved efficiency of cloning of the PCR products generated with Taq DNA polymerase could be achieved by treating the PCR products with a DNA polymerase with 3 ' -5 ' proof-reading exonuclease activity (T4 DNA polymerase, Pfu DNA polymerase etc.) thus removing the overhang dAMP (Costa and Weiner, 1994, Nucl. Acids Res. 22, 2423). The result also shows that 100% of the analyzed clones carried the 420 bp insert, which confirms the positive selection capability of the vector pRGRl Ap. The results also indicate that the other mutations have minimum effect on the function of β-lactamase, and hence Pml I could be used as the unique cloning site. The DNA sequence and restriction sites around the cloning site Pml I is also shown in FIG. 2.

Use of pRGRl Ap Eliminates Exonuclease-induced False Positive Clones A positive selection vector developed based on reconstruction of a reporter gene should greatly reduce, if not eliminate, generation of false positive clones. Thus pRGRlAp was tested for its capability of eliminating false positive clones in a cloning experiment. An aliquot of 1 μg of the vector pRGRlAp was digested with 20 U of Pml I for 1 hr and another aliquot of 1 μg of the vector pRGRlAp was digested with 100 U of Pml I for 4 hr at 37°C. Similarly, an aliquot of 1 μg of the vector pUC19 was digested with 20 U of Sma I for 1 hr and another aliquot of 1 μg of the vector pUC19 was digested with 100 U of Sma I for 4 hr at 25°C. The digests were treated at 70°C for 30 min to inactivate the restriction endonucleases. The digested vectors were then diluted with water (10 ng/μl) and recircularized by ligation, the conditions of which are given below: lμl (10 ng) of vector DNA

4 μl of 5X ligase buffer 14 μl of DEPC-treated water l μl of T4 DNA ligase

Ligation mixture was incubated at 16°C for overnight.

An aliquot of 2 μl of ligation mix was used to transform 50 μl of competent Maxefficiency DH5α E. coli cells for 30 min on ice, followed by heat shock at 42°C for 50 sec, 2 min on ice, addition of 1.0 ml of SOC medium, and incubation at 37°C for 1 hr. For the pUC19 derivatives, an aliquot of 50 μl of the transformation mix was plated onto LB agar plates containing 100 μg/ml ampicillin, 100 ng/ml X-gal and 1 mM IPTG and incubated overnight at 37°C. For the pRGRlAp derivatives, cells from 1 ml transformation mix were spun down, resuspended in 50 μl of S.O.C. medium, and was then plated onto LB agar plates containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline and incubated overnight at 37°C. In case of pUC19 derivatives, all white colonies were considered false positive clones, whereas, the blue colonies were wild-type clones, hi case of pRGRlAp derivatives all clones were considered false positive clones. Table 2 shows the effect of exonuclease digestion for generating false positive clones in a cloning experiment.

TABLE 2

Vector used in Amount of Enzyme Period of Colonies/ml False positive ligation" DNA (μg) (units) incubation (h) transformation clones⁰ m pRGRlAp 1 20 1 4 4 pRGRlAp 1 100 4 7 7 pUC19 1 20 1 90,000 4,400 pUC19 1 100 4 36,000 17,000

The positive selection vector pRGRl Ap and the chromogenic selection vector pUC19 were separately digested with Pml I and Sma I respectively, and after heat inactivation of restriction enzymes each digest (10 ng) was subjected to self-ligation, t^

For pRGRl Ap derivatives, transformants were grown in presence of 100 μg/ml ampicillin and 12.5 μg/ml tetracycline, whereas, for pUC19 derivatives, transformants were grown in presence of 100 μg/ml ampicillin. c hi case of pRGRlAp derivatives, all colonies were considered false positive clones, h case of pUC 19 derivatives, all white colonies were considered false positive clones, whereas, the blue colonies were considered wild-type clones. It is evident form the data in Table 2 that contaminating exonuclease greatly increases the number of false positive clones in case of pUC19, wherein the cloning site in pUC19 is in the chromogenic lacZ gene. This result also shows that exonuclease has very little effect on generating false positive clones in case of pRGRl Ap. The possibility of these false positive clones arising from spontaneous mutations were considered unlikely, because in such case the number of false positive clones should have been similar in case of both vectors.

EXAMPLE 2 Construction of pBR322Δropl

It was decided to construct a positive selection vector with multiple added features. The vector should contain a pUC origin of replication thus giving higher copy number in E. coli compared to pRGRlAp, which is a derivative of pBR322. The vector also should contain an Ml 3 or fl origin of replication to generate single-stranded form of DNA after co-infection with a helper phage phage. Furthermore, the vector should carry a phage promoter around the cloning site for easy in vitro production of RNA probes of the insert DNA.

The rop gene in pBR322 is responsible for inhibition of copy number and deletion of rop is responsible for higher copy number of pUC vectors in E. coli. Thus it was decided to delete the rop gene from pBR322 using PCR-mediated mutagenesis. The following forward PCR primer PUC681F and reverse primer PBR1380R were synthesized. The PCR product generated from pBR322 using these two primers would not contain the rop gene. Forward primer PUC681F:

5'-GTC GCA AGA TCT TGA AAG CTT GCG CTC TTC CGC TTC CTC GCT CAC-3" (45 bases)

Reverse primer PBR1380R:

5'-CTG AGC AGA TCT TAA TCT AGA GTT CTG CCA AGG GTT GGT TTG CGC-3' (45 bases)

The forward primer PUC68 IF carries the restriction sites Bgl II and Hind III at its 5' end. The reverse primer PBR1380R also carries Bgl II and Xba I restriction sites at its 5' end. Recircularization of the PCR product after Bgl II digestion would give the desired plasmid.

The PCR conditions were:

1 ng pBR322 DNA 10^' μM forward primer PUC681F

10 μM reverse primer PBR1380R

0.2 mM dNTPs (equimolar mixture of dATP, dGTP, dCTP and dTTP) 2.5 μl of 10X low salt buffer for Taqplus DNA polymerase. 2.5 U Taqplus DNA polymerase Distilled water making total volume up to 25 μl.

Two drops of mineral oil was added to overlay on the PCR mixture to prevent evaporation during PCR cycling.

The PCR cycle conditions were as follows:

2 min at 94°C, then 15 cycles with: 1 min at 94°C, 1 min at 55°C, 8 min at 72°C; followed by a final extension step of 10 min at 72°C.

To verify the PCR reaction, 5 μl of the PCR product was electrophoresed in 0.8% agarose gel in presence of ethidium bromide for about 1 hr, and the gel was then photographed under UV light using Polaroid Type 667 films. The rest of the PCR amplified DNA was treated with phenol:chloroform:isoamyl alcohol (25:24: 1) and was then precipitated by ethanol. Precipitated DNA was pelleted by centrifugation and the pellet was dried in air. The dried DNA was dissolved in 25 μl of IX Bgl II restriction buffer and then incubated in presence of 20 U of Bgl II restriction endonuclease for 1 hr at 37°C. The digested DNA was electrophoresed in 0.8% agarose gel. The desired DNA band was excised and DNA was purified using Qiaex kit. The purified DNA was used in ligation as per conditions given below:

15 μl of purified DNA

4 μl of 5X ligation buffer l μl (5 U) ofT4 DNA ligase

Ligation was performed for overnight at 16°C. An aliquot of 2 μl ligation mix was used to transform 50 μl Maxefficiency DH5α E. coli host cells according to the recommended protocols. The transformants were then plated onto LB agar plates containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline and incubated at 37°C overnight. Some transformant colonies were individually grown in 5 ml aliquot of LB broth containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline. Small scale plasmid DNA was isolated from each individual clone, and DNA was then digested with 20 U Bgl II restriction endonuclease. Any plasmid DNA carrying a Bgl II cleavage site was further characterized for the presence of other expected restriction endonuclease cleavage sites. One such plasmid carrying expected restriction endonuclease cleavage sites was named as pBR322Δropl and the restriction map of this vector is shown in FIG. 3. With respect to copy number in E. coli the plasmid pBR322Δropl is equivalent to the pUC vectors.

Construction of ρBR322ΔroplM13.2

It was decided to add the Ml 3 origin of replication to pBR322Δropl which would allow generation of single-stranded form of DNA after co-infection with wild-type Ml 3 or fl or another helper phage. The following forward PCR primer PUCl 19.500F and reverse primer PUCl 19.980R were synthesized to amplify the M13 origin region from pUCl 19.

Forward primer PUCl 19.500F:

5'-GGA AGA TCT AAG CTT ACG TCA AAG CAA CCA TAG TAC GCG Gees' (42 bases) Reverse primer PUCl 19.980R:

5'-GGA AGA TCT CCA TAA AAT TGT AAA CGT TAA TAT TTT GTT AAA ATT CGC-3' (48 bases)

The forward primer PUCl 19.500F carries the restriction sites Bgl II and Hind III at its 5' end. The reverse primer PUCl 19.980R also carries a Bgl II restriction site at its 5' end. Ligation of the Bgl II digested PCR product and pBR322Δropl would generate the desired plasmid.

The PCR conditions were:

I ng pUC119 DNA

10 μM forward primer PUCl 19.500F 10 μM reverse primer PUC 119.980R

0.2 niM dNTPs (equimolar mixture of dATP, dGTP, dCTP and dTTP)

2.5 μl of 10X low salt buffer for Taqplus DNA polymerase 2.5 U Taqplus DNA polymerase

Distilled water making total volume up to 25 μl.

Two drops of mineral oil was added to overlay on the PCR mixture to prevent evaporation during PCR cycling. The PCR cycle conditions were as follows:

2 min at 94°C, then 20 cycles with: 1 min at 94°C, 1 min at 55°C, 2 min at 72°C; followed by a final extension step of 5 min at 72°C.

To verify the PCR reaction, 5 μl of the PCR product was electrophoresed in 1.5% agarose gel in presence of ethidium bromide for about 1 hr, and the gel was then photographed under UV light using Polaroid Type 667 films. The rest of the PCR amplified DNA was treated with phenol:chloroform:isoamyl alcohol (25:24: 1) and was then precipitated by ethanol. Precipitated DNA was pelleted by centrifugation and the pellet was dried in air. The dried DNA was dissolved in 25 μl of IX Bgl II restriction buffer and then incubated in presence of 20 U of Bgl II restriction endonuclease for 1 hr at 37°C. The digested DNA was electrophoresed in 1.5% agarose gel. The desired DNA band was excised and DNA was purified using Qiaex kit. The purified DNA was used in ligation. The plasmid pBR322Δropl was also digested with Bgl II and DNA was treated with phenol:chloroform:isoamyl alcohol (25:24: 1) and was then precipitated by ethanol. Precipitated DNA was pelleted by centrifugation and the pellet was dried in air, dissolved in water and then used in ligation in conjunction with the purified PCR product. The conditions of ligation are given below:

10 μl of purified PCR DNA

5 μl of pBR322Δropl DNA

4 μl of 5X ligation buffer 1 μl (5 U) of T4 DNA ligase

Ligation was performed for overnight at 16°C.

An aliquot of 2 μl ligation mix was used to transform 50 μl Maxefficiency DH5α E. coli host cells according to the recommended protocols. The transformants were then plated onto LB agar plates containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline and incubated at 37°C overnight. Some transformant colonies were individually grown in 5 ml aliquot of LB broth containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline. Small scale plasmid DNA was isolated from each individual clone, and DNA was then digested with 20 U Bgl II restriction endonuclease. Any plasmid DNA carrying a 480 bp Bgl II fragment was further characterized for the presence of other expected restriction endonuclease cleavage sites. One such plasmid carrying expected restriction endonuclease cleavage sites was named as pBR322ΔroplM13.2 and the restriction map of this vector is shown in FIG.4. When infected with M13mpl8 the single-stranded form of pBR322ΔroplM13.2 was obtained

Construction of the Positive Selection Vector pRGR2Aρ It was decided to develop a positive selection vector based on reporter gene reconstruction which would be a derivative of pBR322ΔroplM13.2 carrying the features of pRGRl Ap as well as would contain the T7 phage promoter sequence around the cloning site for easy in vitro production of RNA probes of the insert DNA.

The following forward PCR primer RGR1F was synthesized to introduce Pml I, Nar I, EcoR V, Nde I and Cla I sites in the 3' coding region of β-lactamase. Introduction of the unique Pml I restriction site (5'-CACGTG-3') replaces the last (position 286) tryptophan (5'-TGG-3') of β-lactamase with a valine (5'-GTG-3'). The Nar I site in the β-lactamase gene does not change the amino acid sequence of β-lactamase. Introduction of EcoR V restriction site in β-lactamase gene (bla) changes glutamic acid in position 277 into aspartic acid, whereas, the restriction site Nde I changes isoleucine into methionine in position 275, and glutamine into histidine in position 274. The restriction site Cla I in β-lactamase gene (bla) replaces methionine in position 268 with isoleucine. Forward primer RGR1F:

5'-CAATTA CAC GTG CTTAAT CAGTGAGGC GCC GAT ATC AGC CAT ATGTCTATT TCG TTC ATC GAT AGT TGC CTG-3' (96bases)

The following reverse PCR primer RGR2R was synthesized to introduce the T7 phage promoter and restriction sites of Cla I, EcoR V, Nar I, Nde I, Not I and Sfi I for easy extraction of the insert. Repeats of stop codons in all three reading frames were also introduced downstream of the β-lactamase gene. These stop codons would ensure that translation will be prematurely terminated in a recircularized non-recombinant vector, which would have been subjected to some exonuclease digestion. Thus the reporter gene protein will remain functionally inactive in a recircularized. non-recombinant vector and minimize exonuclease-induced false positive clones.

Reverse primer RGR2R:

5'-ATT AAG CAC GTG TAA TTG AAT AAT AGT TGA ATA GTA ATT GAA TAA CTA TAG TGA GTC GTA TTA GGC GCC ATA TGA TAT CGA TGG CCA AAG CGG CCG CTG TCA GAC CAA GTT TAG TCA TAT ATA CTT TAG-3' (129 bases) PCR product generated from pBR322ΔroρlM13.2 using primers RGR1F and RGR2R would be digested with Pml I and subsequent recircularization would give the desired vector. The PCR conditions were:

1 ng pBR322ΔroplM13.2 DNA 10 μM forward primer RGR1F 10 μM reverse primer RGR2R

0.2 mM dNTPs (equimolar mixture of dATP, dGTP, dCTP and dTTP) 2.5 μl of 1 OX low salt buffer for Taqplus DNA polymerase

2.5 U Taqplus DNA polymerase

Distilled water making total volume up to 25 μl.

Two drops of mineral oil was added to overlay on the PCR mixture to prevent evaporation during PCR cycling. The PCR cycle conditions were as follows:

2 min at 94°C, then 15 cycles with: 1 min at 94°C, 1 min at 55°C, 8 min at 72°C; followed by a final extension step of 10 min at 72°C.

To verify the PCR reaction, 5 μl of the PCR product was electrophoresed in 0.8% agarose gel in presence of ethidium bromide for about 1 hr, and the gel was then photographed under UV light using Polaroid Type 667 films. The rest of the PCR amplified DNA was treated with phenol:chloroform:isoamyl alcohol (25:24:1) and was then precipitated by ethanol. Precipitated DNA was pelleted by centrifugation and the pellet was dried in air. The dried DNA was dissolved in 25 μl of IX Pml I restriction buffer and then incubated in presence of 20 U of Pml I restriction endonuclease for 1 hr at 37°C. The digested DNA was electrophoresed in 0.8% agarose gel. The desired DNA band was excised and DNA was purified using Qiaex kit. The purified DNA was used in ligation as per conditions given below: 15 μl of purified DNA

4 μl of 5X ligation buffer l μl (5 U) ofT4 DNA ligase

Ligation was performed for overnight at 16°C. An aliquot of 2 μl ligation mix was used to transform 50 μl Maxefficiency DH5α E. coli host cells according to the recommended protocols. The transformants were then plated onto LB agar plates containing 12.5 μg/ml tetracycline and incubated at 37°C overnight.

Some transformant colonies were individually transferred to LB agar plates containing 100 μg/ml ampicillin as well as to LB agar plates containing 12.5 μg/ml tetracycline. The clones sensitive to ampicillin were then individually grown in 5 ml aliquot of LB broth containing 12.5 μg/ml tetracycline. Small scale plasmid DNA was isolated from each individual clone, and DNA was then digested with 20 U Pml I restriction endonuclease. Any plasmid DNA carrying a Pml I cleavage site was further characterized for the presence of other expected restriction endonuclease cleavage sites. One such plasmid carrying expected restriction endonuclease cleavage sites was named as pRGR2Ap and the restriction map of this vector is shown in FIG. 5. The vector pRGR2Ap is sensitive to ampicillin, and hence carries a dominant negative mutation in the 3' coding region of β- lactamase because of mutation introduced by the restriction sites. FIG. 6 shows the DNA sequence indicating the Pml I cloning site, T7 phage promoter sequence, and other related restriction sites in pRGR2Ap.

Example of Direct Cloning of PCR Product into pRGR2Ap^*

A 420 bp fragment of the lacZ was separately PCR-amplified using Taq DNA polymerase (without 3 '-5' proofreading exonuclease activity) and Pfu DNA polymerase (with 3 ' -5 ' proof reading exonuclease activity) .

Following are the primers used in amplification of the above mentioned 420 bp DNA fragment: Forward primer LC1261RGRF:

5'-TGG TAA GCT TGC GGC CGC AAA GGC CAC AAT TTC ACA CAG GAA ACA GCT ATG-3' (51 bases) Reverse primer LC1680R:

5'-TTT CAT CAA CAT TAA ATG TGA GCG AGT AAC-3' (30 bases)

The PCR conditions were: lμg E. coli DNA

10 μM forward primer (LC1261RGRF)

10 μM reverse primer (LC1680R)

0.2 mM dNTPs (equimolar mixture of dATP, dGTP, dCTP and dTTP) 2.5 μl of 1 OX buffer for Taq or Pfu DNA polymerase

2.5 U Taq or Pfu DNA polymerase

Distilled water making total volume up to 25 μl

Two drops of mineral oil was added to overlaw on the PCR mixture to prevent evaporation during PCR cycling. The PCR cycle conditions were as follows:

2 min at 94°C, then 25 cycles with: 1 min at 94°C, 1 min at 55°C, 2 min at 72°C; followed by a final extension step of 5 min at 72°C.

To verify the PCR reaction, 5 μl of the PCR product was electrophoresed in 1.5% agarose gel in presence of ethidium bromide for about 1 hr, and the gel was then photographed under UV light using Polaroid Type 667 films.

Ligation was performed using 1-200 ng of Pml I digested pRGR2Ap vector DNA, and 1-5 μl of the PCR product. The conditions of a typical ligation experiment is given below:

1 μl (50 ng) of Pml I digested vector pRGR2Ap DNA 2 μl (200ng) of PCR product

4 μl of 5X ligase buffer

12 μl of DEPC-treated water l μl (5 U) ofT4 DNA ligase

Ligation mixture was incubated at room temperature for 5-30 min or at 16°C for overnight.

Different amounts (2-10 μl) of ligation mix were used to transform 10-100 μl of competent Maxefficiency DH5α E. coli cells. In a typical transformation 50 μl of DH5α was transformed with 2 μl of ligation mixture for 30 min on ice, followed by heat shock at 42°C for 50 sec, 2 min on ice, addition of 1.0 ml of SOC medium, and incubation at 37°C for 1 hr. An aliquot of 25-500 μl of the transformation mix was plated onto LB agar plates containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline and incubated overnight at 37°C. As expected, control ligation sample of vector alone gave only a few transformant, whereas, ligation samples of vector plus PCR fragments gave many transformant colonies. Some transformant clones were individually cultured in 5 ml LB broth containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline for overnight at 37°C. Small scale plasmid DNA was isolated using standard alkaline lysis method. The isolated plasmid DNA was characterized by Not I or Sfi I restriction endonuclease digestion. The result of a typical cloning experiment is shown in Table 3.

TABLE 3 pRGR2Ap^a PCR fragment colonies/ml positive clones/

(vector used in (polymerase used) transformation analyzed clones⁰ ligation) mix^b

50 ng 2 μl 340 9/9

(Taq DNA polymerase)

50 ng 2 μl 1170 9/9

(Pfu DNA polymerase)

50 ng 5 -

a The positive selection vector pRGR2Ap was digested with Pml I restriction enzyme, and after heat inactivation of Pml I the vector DNA was used in ligation.

Transformants were grown in presence of 100 [tg/ml ampicillin and 12.5 Rg/ml tetracycline. c The plasmids from the transformants, were digested with either Not I or Sfi I releasing the insert.

As expected, the Table 3 shows that the vector pRGR2Ap is capable of cloning the PCR fragments carrying 5'-TGGTAA-3'at its 5' end as produced by primers LC1261RGRF and LC1680R. Table 3 also shows that the vector pRGR2Ap is capable of cloning both types of PCR products produced by Taq and Pfa DNA polymerases. However, fewer number of clones are obtained in case of cloning of the PCR products obtained by Taq DNA polymerase because in such case only those PCR products of Taq DNA polymerase are ligated which have no dAMP overhang. Improved efficiency of cloning of the PCR products generated with Taq DNA polymerase could be achieved by treating the PCR products with a DNA polymerase with 3'-5' proof-reading exonuclease activity (T4 DNA polymerase, Pfti DNA polymerase etc.) thus removing the overhang dAMP (Costa and Weiner, 1994, Nucl. Acids Res. 22, 2423). The result also shows that 100% of the analyzed clones carried the 420 bp insert, which confirms the positive selection capability of the vector pRGR2Ap. The results also indicate that the other mutations have minimum effect on the function of β-lactamase, and hence Pml I could be used as the unique cloning site. The DNA sequence and restriction sites around the cloning site Pml I is also shown in FIG. 6.

In conclusion, a strategy for developing a positive selection vector system based on reporter gene reconstruction has been established; and wherein such vectors pRGRlAp and pRGR2Ap have been successfully used for direct cloning of PCR generated DNA fragments. Use of pRGRlAp and pRGR2Ap also greatly reduces exonuclease-induced false positive clones in a cloning experiment.

References

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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).

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Claims

CLAΓMS

1. A strategy for developing a positive selection vector based on reconstruction of a reporter gene.

2. A strategy for developing a positive selection vector based on reconstruction of a regulatory gene controlling the expression of a reporter gene.

3. A strategy for developing a chromogenic or fluorogenic selection vector based on reconstruction of the lacZ or a fluorescent protein gene.

4. A strategy for developing a chromogemc or fluorogenic selection vector based on reconstruction of a regulatory gene controlling the expression of the lacZ or a fluorescent protein gene.

5. A cloning vector developed based on claims 1-4 comprises:

(a) an origin of replication;

(b) a selectable marker gene;

(c) a functionally inactive reporter and/or regulatory gene; and (d) a cloning site.

6. A vector as claimed in claim 5, wherein the origin of replication is:

(a) prokaryotic;

(b) eukaryotic; (c) viral;

(d) plasmid pUC;

(e) plasmid pBR322;

(f) phage Ml 3; and

(g) phage, fl.

7. A vector as claimed in claim 5, wherein the selectable marker gene is:

(a) an antibiotic resistance gene;

(b) an essential gene for the growth of the host;

(c) an essential gene for replication and propagation of the vector in the host cell;

(d) the lacZ gene or a part thereof, and

(e) a fluorescent protein gene.

8. A vector as claimed in claim 5, wherein the reporter gene is:

(a) an antibiotic resistance gene;

(b) an essential gene for the growth of the host;

(c) an essential gene for replication and propagation of the vector in the host cell; (d) a toxic gene;

(e) the lacZ gene or a part thereof, and

(f) a fluorescent protein gene.

9. A vector as claimed in claim 5, wherein the reporter and/or regulatory gene: (a) is a coding sequence carrying a dominant mutation at its 3' end,

(b) is a coding sequence carrying a dominant mutation at its 5' end;

(c) carries functional transcriptional and translational regulatory elements; and

(d) does not produce any functional reporter and/or regulatory gene protein upon transformation of a host cell.

10. A vector as claimed in claim 5, wherein the regulatory gene is:

(a) a positive regulatory gene;

(b) a negative regulatory gene; and (c) a bifunctional regulatory gene. - so i l . A vector as claimed in claims 5, wherein the cloning site is:

(a) a unique restriction enzyme cleavage site;

(b) multiple restriction enzymes cleavage sites; and

(c) not a unique restriction enzyme cleavage site.

12. A vector as claimed in claim 5, wherein the vector carries:

(a) a phage promoter around the cloning site;

(b) a viral promoter around the cloning site;

(c) a T7 promoter around the cloning site; (d) a T3 promoter around the cloning site;

(e) an SP6 promoter around the cloning site;

(f) a prokaryotic promoter around the cloning site; and

(g) an eukaryotic promoter around the cloning site.

13. A vector as claimed in claim 5 carries multiple reporter and/or regulatory genes.

14. The vectors pRGRl Ap and pRGR2Ap.

15. A vector as claimed in claim 5, wherein such a vector is used for: (a) cloning DNA fragments generated by polymerase chain reaction (PCR);

(b) cloning appropriate restriction DNA fragments;

(c) cloning appropriate DNA fragments;

(d) diagnostic cloning;

(e) protein production; and (f) gene therapy.

16. A PCR-generated DNA fragment as claimed in claim 15 is the product of a PCR, wherein a primer used in the said PCR carries:

(a) a nucleic acid sequence that upon ligation reconstructs a functional reporter gene; and

(b) a nucleic acid sequence that upon ligation reconstructs a functional regulatory gene.

17. The insertional reconstruction of a reporter and/or regulatory gene as claimed in claims 1-4 is achieved via using:

(a) ligase; (b) recombinase;

(c) topoisomerase;

(d) any enzyme;

(e) no enzyme;

(f) an adaptor and/or a linker; and (g) an adaptor and/or a linker correcting the mutation in the reporter and/or regulatory gene.

18. A kit for cloning of PCR products into a vector as claimed in claim 15 comprises any or a combination of the following: (a) an aliquot of the said vector in the linearized form in a separate container,

(b) an adaptor and/or a linker correcting the mutation in the reporter and/or regulatory gene in a separate container;

(c) an aliquot of DNA ligase in a separate container for ligating the vector DNA with the PCR products; (d) an aliquot of T4 DNA polymerase in a separate container;

(e) an aliquot of T4 polynucleotide kinase in a separate container;

(f) an aliquot of dNTPs in a separate container,

(g) an aliquot of compatible competent host cells in a separate container, wherein the host cell is capable of replication of the recombinant vector upon transformation;

(h) an aliquot of a control target DNA in a separate container, wherein the said DNA is used for control PCR, the product of which is then ligated as a control into the said vector;

(i) an aliquot of each of forward and reverse control primers in separate containers for PCR amplification of the said target DNA; and

(j) an aliquot of a non-linearized control plasmid in a separate container, wherein the said plasmid is used in a control transformation.