WO2000046366A1 - Isolation of nucleic acid molecules - Google Patents

Abstract

The invention relates to methods and kits for isolating or purifying single or double stranded vectors. Such isolated/purified vectors are obtained through the interaction of all or a portion of functional sequences of the vectors of interest with one or more oligonucleotides which target and bind to one or more of the funcitonal sequences. The isolated/purified vectors produced may be further manipulated or processed by well known molecular biology techniques such as sequencing, digestion, ligation, transformation, amplification and the like.

Description

Isolation of Nucleic Acid Molecules

Background of the Invention

Field of the Invention

The invention relates to nucleic acid purification and/or isolation. The invention also concerns further processing such isolated or purified nucleic acid molecules (e.g. DNA and RNA) by well known molecular biology techniques such as sequencing, digestion, amplification, transformation, and/or synthesis. In particularly, the invention relates to isolation and/or purification of single and/or double stranded vectors, preferably DNA vectors.

Related Art

The first triple-helical structure of nucleic acids was discovered more than 30 years ago (Felsenfeld, G., et al. (1957) J. Am. Chem. Soc. 79:2023- 2024). While the biological roles of such structures are still open to question, their chemical characteristics have been considerably elucidated in recent works (for review, see Wells, R. D., et al. (1988) FASEB J. 2:2939-2949). The most well-characterized triplex is the one formed between a double- stranded homopurine-homopyrimidine helix and a single-stranded homopyrimidine tract. In this type of triple-helix, the third homopyrimidine strand binds to the major groove, parallel to the homopurine strand of Watson- Crick double-helical DNA, via Hoogsteen hydrogen bonding. The third- strand thymine (T) recognizes adenine-thymine (AT) base pairs forming T-A- T triplets, and the third-strand cytosine (C), protonated at its N-3 position, recognizes guanine-cytosine (G-C) base pairs forming C-G-C triplets.

Typically, specific DNA is isolated from heterogeneous DNA mixtures using conventional hybridization based methods (e.g., colony or plaque hybridization (Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor, Cold Spring Harbor Press). These well-established methods while quite reliable have some practical drawbacks. First, they require time-consuming and labor-intensive steps of filter preparation that often limit the number of clones that can be screened. Furthermore, since these procedures include prior denaturation steps and other treatments that destroy the integrity of the target DNA molecules, one has to re-isolate the corresponding clones from the original plates to obtain intact DNA molecules for further biological biochemical manipulations. Third, sequences toxic to the host sometimes hamper successful cloning. Fourth, the natural modifications of the target DNA are not maintained during cloning. Finally, despite recent development of Yeast Artificial Chromosome (YAC) and Bacterial Artificial Chromosomes (BAC) vectors, it is still difficult to clone very large DNAs. Obviously, non-cloning-based biochemical methods to isolate specific DNA from a complex mixture would be of some help with these problems. However, such methods are still not satisfactory. Biochemical purification by density and size fractionation after cleavage with restriction enzymes can be applied only in limited instances (Tsujimoto, Y. and Suzuki, Y. (1984) Proc. Natl. Acad. Sci. USA 81 :1644- 1648). The polymerase chain reaction (PCR) fulfills some of these needs and provides large amounts of DNAs (Mullis, K. B. and Falocna, F. A. (1987) Methods Enzymol 155:335-350), but it is typically limited to relatively short (<10 kb) DNA fragments. Furthermore, natural modifications of the original DNA cannot be maintained. Although an effective method of affinity chromatography for DNAs was reported (Tsurui, H. et al. (1990) Gene 88:233-239), it requires the prior denaturation of target DNA molecules and elution by denaturation.

Several screening procedures, potentially applicable to large DNAs, that keep the target DNA in its native double stranded form have been developed using RecA protein (Honigberg, S. M., et al. (1986) Proc. Natl. Acad. Sci. USA 83:9586-9590; Rigas, B., et al. (1986) Proc. Natl. Acad. Sci. USA 83:9591-9595). An affinity capture procedure using hybridization at the end of a large DNA fragment was also reported (Kandpal, R., et al. (1990) Nucleic Acids Res. 18:1789-1795). However, these procedures include the handling of DNAs in solution, in at least several steps, which inevitably breaks large DNAs into smaller pieces. Cantor et al. (U.S. Patent No. 5,482,836) and Wang et al. (U. S. Patent

No. 5,401,632) developed methods to capture double stranded plasmids using triple helix affinity. However, these methods rely on specialized plasmids which have been engineered to contain target sequences to allow triple helix formation.

Summary of the Invention

The present invention provides an improved method for isolating and/or purifying nucleic acid molecules from a sample (preferably a biological sample) by forming a complex between a targeting oligonucleotide and the target nucleic acid and separating the complex from the sample. The target nucleic acid molecule may then be recovered from the complex. Unlike previous methods, the present invention provides a method to generally target nucleic acid molecules (preferably double or single stranded vectors and most preferably double stranded DNA vectors) without the need to add or incorporate a target sequence to the nucleic acid molecule of interest. The invention thus allows isolation/purification of broad classes or groups of molecules or vectors commonly used in the field of molecular biology without the need to construct specialized molecules or vectors. The present invention thus provides a method for isolating and/or purifying single or double stranded vectors (preferably DNA vectors) in a sample. In a preferred aspect, the separation of vectors from the sample relies on a support, preferably a solid or semi-solid support. In accordance with the invention, a sample is contacted with one or more oligonucleotides specific for one or more target vectors. Preferably, the oligonucleotide(s) are coupled either directly or indirectly to one or more haptens. The haptenylated oligonucleotides-sample mixture is incubated under sufficient conditions to allow binding of the oligonucleotides with the particular target vector in the sample. The reaction mixture containing the vector-oligonucleotide complex is preferably contacted with a support to which one or more ligands are directly or indirectly bound. Interaction between the haptens of the oligonucleotides and the ligands of the support allow the vector- oligonucleotide complex to be bound to the support. In another aspect, the oligonucleotides may be directly bound to the support thereby avoiding the need to use ligand/hapten interactions. The support bearing the complex is then separated from the contaminants in the reaction mixture/sample by, for example, washing the support with an appropriate solution (e.g., buffers, water, etc.). The isolated/purified vectors may then be obtained by removing the vector from the support. Preferably, the vectors are separated from the support by treating the mixture with a reagent that breaks or dissociates the interaction between the oligonucleotides and the vectors without adversely affecting the single or double stranded vectors to be isolated and/or purified. Alternatively, vectors may be removed from the support by breaking or dissociating the interaction between the haptens of the oligonucleotide and the ligand of the support. The particular vectors of interest are then recovered. In a preferred aspect, the invention can be used to isolate/purify vector populations such as genomic or cDNA libraries.

In another aspect of the invention, the invention provides purified and/or isolated vectors which may be further manipulated by standard recombinant DNA techniques. Such techniques include but are not limited to amplification, transformation, digestion, sequencing and the like.

In specific embodiments of the invention, the one or more oligonucleotides are specific for target sequences (or portions thereof) which are common to vectors. Such target sequences typically are functional sequences which impart useful phenotypic or genotypic characteristics to the vectors or host cells containing the vectors. Thus, because the target sequences (or portions thereof) of the vectors are functional sequences typically found in many groups or classes of vectors, the invention provides a general method to isolate/purify any number of vectors having such common functional sequences. Such sequences (or portions thereof) include but are not limited to origins of replication, promoters, marker or selection genes or sequences, antibiotic resistance genes or sequences, indicator genes or sequences, repressor sequences, primer sequences, multiple cloning site sequences, terminator sequences, transcription sequences, translation sequences, tag sequences, recombination sequences, and portions thereof.

In accordance with the invention, the hapten-ligand system for isolating/purifying the vector(s) of interest can be any one or more haptens that binds with its corresponding one or more ligands. Such systems will be readily recognized by those skilled in the art and include antigen/antibody, an avidin/biotin, a streptavidin/biotin, a protein A/Ig and a lectin/carbohydrate system. In accordance with the invention, the support will be any support to which may be bound one or more ligands for isolating and/or purifying the vectors of interest. Such supports may be solid supports or semi-solid supports, but are preferably solid supports. Supports for the invention may be prepared using any number of materials including plastic, glass, agarose, metal, nitrocellulose, acrylamide, silica, nylon, cellulose, diazocellulose, polystyrene, polyvinyl chloride, polypropyline, polyethylene, dextran, polydirinyl fluoride, sepharose, polyacrylamide, polystyrene divinyl benzene, polyvinyltoluene, modified polystyrene, polysaccharides, acrylic polymers, hydroxylapatite, agar, starch, nylon, and latex. The form of such supports may vary from beads, particles, filters, columns, microtiter plates and the like. Moreover, the supports of the invention may be magnetic, paramagnetic, or superparamagnetic. In a preferred aspect, the support are beads (preferably magnetic, paramagnetic or superparamagnetic beads).

The present invention also provides kits for isolating and/or purifying vectors of interest. Such kits may comprise any one or a number of components for carrying out the methods of the invention including one or more oligonucleotides of the invention, one or more of the supports of the invention, one or more solution or buffers for removing contaminants (e.g. wash solutions) and one or more solutions for removing the vectors of interest from the support material. Such kits may also comprise additional components for further manipulation or processing the isolated/purified vectors.

The invention also relates to a composition comprising a triple stranded complex comprising a single stranded oligonucleotide(s) of the invention and a double stranded vector. The invention also concerns compositions comprising double stranded complexes comprising a single stranded oligonucleotide of the invention and a single stranded vector. Where two or more oligonucleotides are used to target the same target in the vector of interest, the compositions of the invention may comprise triple stranded complexes (e.g. two oligonucleotides and the single stranded vector), four stranded complexes (e.g. two oligonucleotides and a double stranded vector), etc. In a preferred aspect, the oligonucleotides are bound (directly or indirectly) to one or more haptens or may be bound (directly or indirectly) to one or more supports. The compositions may also comprise a support which contains one or more ligands which are capable of binding to the haptenylated oligonucleotides.

Brief Description of the Figures

Figure 1 shows the isolation of plasmid DNA through the formation of a triple stranded complex. Lane M: 1 kb DNA ladder; Lane 1 and 2: no oligo was added; Lane 3 and 4: oligo Trip 1 was added; Lane 5 and 6: oligo Trip 2 wad added; and Lane 7 and 8: oligo Trip 3 was added.

Figure 2 shows the comparison of plasmid DNA isolation from cell crude extract. Lane M: 1 kb DNA ladder; Lane 1 : the DNA was isolated with the kit of Concert Rapid Plasmid Miniprep System; Lane 2: the DNA was isolated with the kit of Concert Rapid Plasmid Miniprep System followed by the purification of triplex formation; Lane 3: the DNA was isolated with the protocol of lysozyme/boiling lysis; and Lane 4: the DNA was isolated with lysozyme/boiling lysis followed by the purification of triplex formation.

Detailed Description of the Preferred Embodiments

In the description that follows, a number of terms used in the fields of molecular biology and recombinant DNA technology are utilized extensively. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms_> the following definitions are provided.

Amplification. As used herein, "amplification" refers to any in vitro method for increasing the number of copies of a nucleotide sequence with the use of a polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a nucleic acid (e.g., DNA) molecule or primer thereby forming a new nucleic acid molecule complementary to the nucleic acid template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of nucleic acid synthesis. Amplification reactions include, for example, polymerase chain reactions (PCR). One PCR reaction may consist of 5 to 100 "cycles" of denaturation and synthesis of a nucleic acid molecule.

Gene. A DNA sequence that contains information necessary for expression of a polypeptide or protein. It includes the promoter and the structural gene as well as other sequences involved in expression of the protein.

Bound. As used herein "bind", "bound", or other similarly used terms refer to both covalent and non-covalent associations and/or interactions, but can also include other molecular associations such as Hoogsteen hydrogen bonding and Watson-Crick hydrogen bonding. Hybridization. The terms "hybridization" and "hybridizing" refers to the pairing of two complementary single-stranded nucleic acid molecules (RNA and/or DNA) to give a double-stranded molecule. As used herein, two nucleic acid molecules may be hybridized, although the base pairing is not completely complementary. Accordingly, mismatched bases do not prevent hybridization of two nucleic acid molecules provided that appropriate conditions, well known in the art, are used.

Host. Any prokaryotic or eukaryotic cell that is the recipient of a vector. The terms "host" or "host cell" may be used interchangeably herein. For examples of such hosts, see Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). Preferred prokaryotic hosts include, but are not limited to, bacteria of the genus Escherichia (e.g. E. coli), Bacillus, Staphylococcus, Agrobacter (e.g. A. tumefaciens), Streptomyces, Pseudomonas, Salmonella, Serratia, Caryophanon, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest in the present invention include E. coli K12, DH10B, DH5α and HB101. Preferred eukaryotic hosts include, but are not limited to, fungi, fish cells, yeast cells, plant cells and animal cells. Particularly preferred animal cells are insect cells such as Drosophila cells, Spodoptera Sf9 and Sf21 cells and Trichoplusa High-Five cells; nematode cells such as C. elegans cells; and mammalian cells such as COS cells, CHO cells, VΕRO cells, 293 cells, PΕRC6 cells, BHK cells and human cells.

Nucleotide. As used herein "nucleotide" refers to a base-sugar- phosphate combination. Nucleotides are monomeric units of a nucleic acid sequence (DNA and RNA). The term nucleotide includes ribonucleoside triphosphates such as ATP, CTP, UTP, GTP, deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP and any methylated ATP, CTP, UTP, GTP, dATP, dCTP, dITP, dUTP, dGTP and dTTP. The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP. According to the present invention, a "nucleotide" may be unlabeled or detectably labeled by well known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, biotin labels and enzyme labels.

Oligonucleotide. As used herein "oligonucleotide" refers to a chain of two or more nucleotides although derivative oligonucleotides such as peptide nucleic acid (PNA) oligonucleotides, morpholino oligonucleotides, pyrrole- imidazole polyamides and the like are contemplated by this term.

Vector. A vector is a single or double stranded nucleic acid molecule (preferably DNA, preferably double stranded and/or preferably circular) capable of replicating autonomously in a host cell. Such vectors may also be characterized by having a small number of endonuclease restriction sites at which such sequences may be cut without loss of an essential biological function and into which nucleic acid molecules may be spliced to bring about its replication and cloning. Thus, the vectors may or may not contain sequences of genes or other sequences of interest (e.g. recombinant vectors). Examples include plasmids, autonomously replicating sequences (ARS), centromeres, cosmids and phagemids. Vectors can further provide primer sites, e.g., for PCR, transcriptional and/or translational initiation and/or regulation sites, recombinational signals or sites, replicons, etc. The vector can further contain one or more selectable markers suitable for use in the identification of cells transformed or transfected with the vector, such as kanamycin, tetracycline, ampicillin, etc.

In accordance with the invention, any vector may be used. In particular, vectors known in the art and those commercially available (and variants or derivatives thereof) may be used in accordance with the invention. Such vectors may be obtained from, for example, Vector Laboratories Inc., InVitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Amersham Pharmacia Biotech, EpiCenter, OriGenes Technologies Inc., Stratagene, Perkin Elmer, Pharmingen, Life Technologies, Inc., and Research Genetics. Such vectors may then, for example, be used for cloning or subcloning nucleic acid molecules of interest. General classes of vectors of particular interest include prokaryotic and/or eukaryotic cloning vectors, expression vectors, recombinational cloning vector(s), fusion vectors, two- hybrid or reverse two-hybrid vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts (e.g., PACs YACs and BACs) and the like.

Other vectors of interest include viral origin vectors (Ml 3 and fl vectors, bacterial phage λ vectors, baculovirus vectors, adenovirus vectors, and retrovirus vectors), high, low and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (pACYC184 and pBR322) and eukaryotic episomal replication vectors (pCDM8). Particular vectors of interest include prokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Inc.), pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Amersham Pharmacia Biotech, Inc.), pKK233-2 and pKK388-l (Clontech, Inc.), and pProEx-HT (Life Technologies, Inc.) and variants and derivatives thereof. Vectors can also be eukaryotic expression vectors such as pFastBac, pFastBac HT, pFastBac DUAL, pSFV, and pTet-Splice (Life Technologies, Inc.), pEUK-Cl, pPUR, pMAM, pMAMneo, pBHOl, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHl lO, and pKK232-8 (Amersham Pharmacia Biotech, Inc.), p3'SS, pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBsueBacIII, pCDM8, pcDNAl, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Inc.) and variants or derivatives thereof. Other vectors of particular interest include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, fosmids (pFOSl), YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), pBAC108L, pBACe3.6, pBeloBACl 1 (Research Genetics), PACs, PI (E coli phage), pQΕ70, pQE60, pQE9 (Qiagen), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (InVitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Amersham Pharmacia Biotech), pSPORTl, pSPORT2, pCMVSPORT2.0, pSV-SPORTl (Life Technologies, Inc.), and the vectors described in Provisional Patent Application No. 60/065,930, filed October 24, 1997, and WO99/21977, the entire contents of which are herein incorporated by reference, and variants or derivatives thereof.

Two-hybrid and reverse two-hybrid vectors of particular interest include pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGADl-3, pGADIO, pACt, pACT2, pGADGL, pGADGH, pAS2-l, pGAD424, pGBT8, pGBT9, pGAD- GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof.

Examples of target sequences contained by vectors which may be used in the isolation/purification procedures of the invention include any sequence or portion thereof commonly found in a vector. Such sequences are sequences that have not been used to isolate/purify the vector. Preferably, such sequences are functional sequences in that they impart a phenotypic and/or genotypic characteristic to the vector or to the host containing the vector or to the product or protein (peptide) encoded by the vector. For example, target sequences include but are not limited to any gene or portion thereof associated with a vector such as antibiotic resistance genes or portions thereof (e.g. tetracycline, ampicillin, neomycin, kanamycin, chloroamphenicol etc.), reporter or marker genes or portions thereof (e.g., β-galactosidase gene, luciferase gene, lacZ, green fluorescent protein, chloramphenicol transferase), suicide and/or death genes or portions thereof (e.g., ccdB lethal gene, Dpnl methylase, sucB (sucrose sensitivity), streptomycin L and restriction enzyme genes such as Bam HI, Eco RI, Hae III etc.), any gene encoding a particular protein/peptide of interest or portions thereof, any termination sequences or portions thereof, any primer sequence or portions thereof (such as standard sequencing Ml 3 forward or Ml 3 reverse primers), any multiple cloning site sequences or portions thereof, any translational functional sequences or portion thereof (such as cap structure (m⁷GpppN), Shine-Dalgarno (SD) sequence, poly(A) and poly (dA) tails), any transcriptional functional sequences or portion thereof (such as promoters, polyadenylation signals, splice sites, enhancer sequences), any replication sequences or portions thereof (such as plasmid origins of replication, viral origins of replication, eukaryotic replication origins, FI intergenic region), any protein tag sequences or portions thereof (such as GST, His Tag, MBP, ZZ, pinpoint, strep-tag, Flag peptide), and any recombination sequences or portions thereof such as attB, attL, attP, attR, loxP, (and mutants or derivatives thereof) and those described in PCT application WO 96/40724.

Since the sequences of many vectors are known, one can easily select the appropriate target sequences for oligonucleotide design in accordance with the invention. Alternatively, the vectors can easily be sequenced by standard techniques so that appropriate targets can be selected. Conditions for forming the oligonucleotide-nucleic acid target complex and the length of the oligonucleotide may vary depending on the target sequence used. Such conditions and the type and length of the oligonucleotide may be determined and optimized using standard binding assays such as the transformation or gel assay described in the Examples. For isolating double stranded vector targets, an oligonucleotide which is pyrimidine rich is preferably used to bind purine- pyrimidine rich target sequence, thereby forming a triple stranded molecule. For sequences that consist of purine nucleotides, nucleotide derivatives such as deoxyinosine (I), 9-(l-β-D-2-deoxyribofuranosyl)-2-amino-6- methoxyaminopurine (dK), and/or 3-nitropyrrole deoxyribonucleoside (M or dM) can be used to construct the oligonucleotides of the invention to allow for more efficient binding of the oligonucleotide to the double stranded vector. Other such derivatives for use in the invention will be readily apparent to one skilled in the art. Thus, the invention relates to such derivative oligonucleotides used in the methods of the invention.

A list of base analogues which could substitute the base adenine and guanine in capture sequence include but are not limited to:

Table 1 shows a number of oligonucleotide sequences specific for particular vector target regions including origins of replication, intergenic regions and gene sequences. Table 2 shows a number of oligonucleotide sequences for targeting various vector antibiotic resistance genes.

Table 1

Table 2

Other target sequences of interest commonly found in vectors include FI intergenic region, replication origins, lac I, lac Z, lac A, lac Y, antibiotic resistance genes, luciferase genes, and SV40 intron and polyadenylation signals. In accordance with the invention, one or more oligonucleotides can be prepared to target such sequences or portions thereof.

Examples of vectors containing FI intergenic region include: Yeast integrative vector, Cloning vector pALTER-1, Cloning vector pGEM-5Zf(+), pBluescript II SK(+) vector DNA, pBluescript II KS(+) vector DNA, Yeast episomal vector pRS426, Yeast integrative vector pRS305, pICEM19R minus plasmid cloning vector, Cloning vector pGEM-13Zf(+), Plasmid pKAl DNA, Yeast centromere vector pRS414, Yeast centromere vector pRS413, pEMBL 9 minus phasmid cloning vector, pEMBL 8 minus phasmid cloning vector, Yeast centromere vector pRS415, Cloning vector pEMBL 8 minus (pEMBLδm), Cloning vector pGEM-7Zf(+), Yeast centromere vector pRS416, pBluescript SK(+) vector DNA, Yeast centromere vector pRS314, pBluescript KS(+) vector DNA, BlueScribe KS Plus cloning vector, and the like, and variants or derivatives thereof. Examples of vector containing replication origin sequence include:

Cloning vector pLXSH, Cloning vector pGEMEX-2, pSP6T719 cloning vector, pJSC73 cloning vector from pBR325 and pAT153, multicopy Saccharomyces cerevisiae/E. coli shuttle vector, YΕp24 yeast extrachromosomal plasmid, pMC1511 cloning vector, pEX3 expression vector, Plasmid pKUN9, a cloning vector, Synthetic clomng vector plasmid pHSG664, Cloning vector cosmid pTCF, Yeast episomal vector pRS425, Cloning vector pDR2, pFNeo eukaryotic expression vector, E. coli plasmid synthetic cloning vector pET31FlP, Yeast centromere vector pRS315, and the like, and variants or derivatives thereof. Examples of vectors that contain the lac I, lac Z, lac Y and lac A genes include: Cloning vector pFRT2, M13mpl l phage cloning vector, M13mpl0 phage cloning vector, Integrational vector pMUTIN2, pUR290 cloning vector, Cloning vector pNASSbeta, Cloning vector M13mpl8, M13mp9 phage cloning vector, Cloning vector pPD21.28, expression vector pUEX2, pGEX- 6P-3 cloning vector, plasmid pLGlacz7, pCHl lO cloning vector, pGEX-2TK cloning vector, Cloning vector pCMVbeta, transposon Tn5-OT182, Cloning vector pPD16.43, Cloning vector pLacZi, pUR291 cloning vector, Cloning vector pTL61T, Cloning vector pGlac, and the like, and variants or derivatives thereof. Examples of vectors that contain antibiotic resistance genes include:

Shuttle vector pHY320PLK DNA, Cloning vector pEG202 (pLexA), Cloning vector pGL2-Promoter, Cloning vector pGEM-4Z, BlueScribe cloning vector, Cloning vector pKK388-l, Plasmid pUT18, Plasmid pH2515, shuttle vector, Reporter vector pCRE-Luc, Cloning vector pJG4-5 (pB42AD), Cloning vector pTRE, Cloning vector pDG1731, Ligation-independent cloning vector pBluescript, pKK232-8 cloning vector, pDR540 cloning vector, Cloning vector pGEM-13Zf(+), Fusion cloning vector pTRXFUS, Cloning vector pADGal4 2.1, Expression vector pNEX, Yeast CUP1 expression/integration cloning vector, Cloning vector pKIL109, Cloning vector pKIL108, and variants or derivatives thereof. Examples of vectors that contain luciferase genes include: Eukaryotic luciferase expression vector pCMVtkLUC+, Cloning vector pGL3-Promoter, Cloning vector pSP-luc+NF, Eukaryotic luciferase expression vector pLUC+, Cloning vector pGL3 -Enhancer, Cloning vector pVLH-1, Clomng vector pGL3-Basic, Eukaryotic luciferase expression vector ptkLUC+, Cloning vector pGL3-Control, Cloning vector pSP-luc+, Eukaryotic luciferase expression vector pTATALUC+, Reporter vector p21uc, Cloning vector pFR- Luc, Expression vector pBSII-LUCIN, Expression vector pZElPAllacO-1 luc, Cloning vector pGL2-Basic, Cloning vector pMAMneo-LUC, Cloning vector pGL2-Control, and variants or derivatives thereof. Examples of vectors that contain SV40 intron and polyadenylation signals include: Cloning vector pFAC-dbd, Signal sequence detection vector pSSD3, Cloning vector pEGFP-N2, Expression vector pNEX, Cloning vector pMAMneoBlue, Expression vector pVP-HAl, Cloning vector pSV2neo, Cloning vector pCMVTAG4a, Epitope tagging vector pCMV-Tag 1 , Cloning vector pSEAP-Control, Ligation-independent promoter-cloning vector, Reporter vector pSRF-Luc, Cloning vector pBI-GL, Cloning vector pMUT- Elk, Cloning vector pFA2-elkl, Co-reporter vector pRL-SV40, Expression vector pNEX delta, Cloning vector pCMVTAG5a, Cloning vector pCMV- scriptEX, Reporter vector pCAT3 -Enhancer vector, and variants or derivatives thereof. Isolation of Nucleic Acid Molecules

According to the present methods, single- or double-stranded nucleic acid molecules (preferably vectors and most preferably DNA vectors) are isolated and/or purified. Such molecules are rapidly isolated from solution by binding the nucleic acid molecules to a support. According to the invention, vectors are isolated/purified through binding of one or more oligonucleotides to one or more target sequences on a vector of interest. In this way, the nucleic acid molecules of interest can be isolated/purified from any sample or reaction mixture.

The vectors can be RNA or DNA vectors, double stranded or single stranded, circular or linear, or any combination thereof. When the vectors are single stranded, the oligonucleotide will under sufficient conditions bind or hybridize to the single stranded vector forming a double stranded molecule where the oligonucleotide binds to the vector target sequence. When the vectors are double stranded, the oligonucleotide under appropriate conditions binds to the double stranded vector forming a triple stranded molecule where the oligonucleotide binds to the vector target sequence. In another aspect, multiple oligonucleotides specific for a single target site may bind to the single or double stranded vectors thereby forming triple stranded, four stranded etc. complexes. Preferably, the invention is used to isolate/purify double stranded vectors. In another aspect, two, three, four, five or more oligonucleotides are used to target the same or two, three, four, five or more different target vector sequences. Such multiple oligonucleotide/multiple target sequences may be used to isolate the same or different vectors and may be used sequentially or at the same time. Thus, the invention allows isolation/purification of a number of different vectors using different targets from a sample. Alternatively, increased selectivity or purity of a single type of vector may be achieved using a number of different target sequences for the single vector of interest. Moreover, multiple haptens may be used in combination with multiple types of oligonucleotides or a single type of oligonucleotide to allow increased binding or higher capacity for binding the oligonucleotide-vector to the support. Likewise, multiple different ligands may be used on a support or multiple supports each having different ligands may be used in the invention. Thus, use of multiple supports may provide a means to isolate/purify multiple vectors from a sample.

In the practice of the invention, any support may be used to bind the oligonucleotides or to bind hapten-specific ligand molecules. Preferably, solid supports are used. Preferred such solid supports include, but are not limited to nitrocellulose, diazocellulose, glass, silica, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads and microtitre plates. Preferred are beads made of glass, latex or a magnetic material, and particularly preferred are magnetic, paramagnetic or superparamagnetic beads. Linkage of the ligand molecule or the oligonucleotide to the support can be accomplished by any method such as covalent, non-covalent, hydrophobic or ionic coupling (including coating) that will be familiar to one of ordinary skill in the art.

According to the invention, any hapten molecule having the capability of binding the ligand molecule may be used. Particularly preferred hapten ligand molecules for use in the invention include without limitation: (i) avidin and streptavidin; (ii) protein A, protein G, a cell-surface Fc receptor or an antibody-specific antigen; (iii) an enzyme-specific substrate; (iv) polymyxin B or endotoxin-neutralizing protein (ENP); (v) Fe ^^1"; (vi) a transferrin receptor; (vii) an insulin receptor; (viii) a cytokine (e.g., growth factor, interleukin or colony-stimulating factor) receptor (ix) CD4; (x) spectrin or fodrin; (xi) ICAM-1 or ICAM-2; (xii) C3bi, fibroinogen or Factor X; (xiii) ankyrin; (xiv) integrins o-iβ], α₂βι, α₃βι, α₆β_l5 α β_l5 and α₆β₅; (xv) integrains α₃βι, α₄β_1? α₄β₇, α₅β_l5 α_vβι, αubβ₃, α_vβ₃; and α_vβ₆; (xvii) integrins α_vβι and α_vβ₃; (xviii) vitronectin; (xix) fibronectin; (xx) collagen; (xxi) laminin; (xxii) glycophorin; (xxiii) Mac-1; (xxiv) LFA-1; (xxv) β-actin; (xxvi) gpl20; (xxvii) cytokines (growth factors, interleukins or colony-stimulating factors); (xxviii) insulin; (xxix) ferrotransferrin; (xxx) apotransferrin; (xxxi) lipopolysaccharide; (xxxii) an enzyme; (xxxiii) an antibody; and (xxxiv) biotin. Binding of the hapten(s) to the oligonucleotide of the invention may be accomplished using techniques well known in the art.

For example, in a preferred aspect of the invention where the ligand is biotin, a biotin-binding hapten such as avidin or streptavidin may be linked to the oligonucleotide. Alternatively, the support ligand may be avidin or streptavidin and the oligonucleotide hapten may be biotin. In a particularly preferred such aspect, the solid support used are avidin- or streptavidin- coupled magnetic, paramagnetic or superparamagnetic beads which are commercially available, for example, from Dynal A.S. (Oslo, Norway), Seradyne (Indianapolis, IN) or from Sigma (St. Louis, Missouri). Of course, the choice of ligand will depend upon the choice of hapten used; appropriate ligands for use in the methods of the invention will thus be familiar to one of ordinary skill in the art. To isolate the nucleic acid molecules produced by the methods of the invention, the solution or sample comprising the vector complex is contacted with the one or more oligonucleotides under conditions favoring binding. The oligonucleotides may be bound directly to one or more haptens which may be bound to one or more supports through ligand-hapten interactions. In a preferred aspect, host cells containing the vector(s) of interest are lysed using well known techniques such as chemical, enzymatic or mechanical lysis and the crude extract containing the vector(s) are contacted with the haptenylated oligonucleotide(s) and the ligand-coupled support(s) or the oligonucleotide(s) coupled support(s). Alternatively, reaction mixtures in which the vectors have been manipulated (e.g. digestion, amplification, ligation etc.) may be used as a sample to isolate/purify vector(s). Typically, conditions for contacting the vectors with oligonucleotide(s) and with the support include incubation in the presence of buffered salt solutions, preferably a TRIS-, phosphate-, HEPES- or carbonate-buffered sodium chloride solution, more preferably a TRIS- buffered sodium chloride solution, still more preferably a solution comprising about 10-100 mM TRIS-HC1 and about 300-2000 mM NaCl, and most preferably a solution comprising about 0.1M sodium phosphate buffer and about 2 M NaCl, at a pH of about 4-9, more preferably a pH of about 4-8, still more preferably a pH of about 4.5-6.5, and most preferably a pH of about 6.0. Incubation is preferably conducted at 0°C to about 60°C, and most preferably at about 50°C, for about 30-240 minutes, preferably about 45-120 minutes, and most preferably about 120 minutes.

Once the one or more vectors have been bound to the support, unwanted or contaminant materials (such as buffers, enzymes, proteins, nucleases, cells and cell debris, and contaminating nucleic acid molecules such as chromosomal DNA molecules, RNA molecules, nucleotides, etc.) may be eliminated by simply removing them in the supernatants. For example, in a preferred aspect in which the vectors of interest are bound to beads or other particulate support, the mixture containing the vectors is mixed and incubated for a sufficient time and under sufficient conditions to bind the vectors to the beads or support and then the vector-bead complex is separated from the mixture by any physical means (e.g. filtering, centrifugation, magnetic means, etc.) and any remaining supernatant containing the contaminants may be removed by aspiration, pipetting etc. The vector-support or vector-bead complex may then be washed any number of times with any solution compatible with the vector complexes of interest (preferably one or more buffers) to further remove contaminating materials. In a particularly preferred aspect, ligand-coupled magnetic, paramagnetic or superparamagnetic beads are used as the support and the vector-beads complex is segregated from the supernatants using a magnet (such as a Magna-Sep Magnetic Particle Separator; Life Technologies, Inc.). Prior to their release from the support, the immobilized vectors are preferably washed one or more times, for example with one of the buffered salt solutions described above, to more fully remove unwanted materials.

Once the contaminants have been substantially or fully removed, the vector molecules may be released from the support by contacting the support with any solution which may dissociate or remove the vector(s) from the oligonucleotide(s) and/or dissociate the hapten-ligand interaction. Preferred conditions for release of the vectors from the support include increasing temperature, changing pH, and/or changing salt concentrations. Preferably, such release is accomplished by changing pH. Following their release from the support, the vectors may be further processed or manipulated by techniques that are well-known in the literature such as digestion, ligation, amplification, nucleic acid synthesis, transformation into one or more host cells and sequencing (see e.g., Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, 2ⁿ ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp. 8.60-8.63 (1987)), and others that will be familiar to one of ordinary skill in the art. Particularly preferred amplification methods according to this aspect of the invention include PCR (U.S. Patent Nos. 4,683,195 and 4,683,202), Stranded Displacement Amplification (SDA; U.S. Patent No. 5,455,166; EP 0 684 315), and Nucleic Acid Sequence-Based Amplification (NASBA; U.S. Patent No. 5,409,818; EP 0 329 822). Most preferred are those methods comprising one or more PCR amplifications.

Kits

The present invention also provides kits for use in isolation and/or purification of the vectors of interest and optionally for further manipulation or processing of the isolated/purified vectors. Kits according to this aspect of the invention comprise a carrier such as a box, carton, tube or the like, having in close confinement therein one or more containers, such as vials, tubes, ampules, bottles, microtiter plates, and the like, wherein the kit contains one or more components selected from the group consisting of one or more oligonucleotide(s) of the invention (which are preferably haptenylated), one or more supports or ligand-coupled supports, one or more wash buffers to remove unwanted contaminants and one or more release buffers to remove the vectors from the support. In additional aspects, the kits of the invention may further comprise instructions for isolating/purifying the vectors of interest and other reagents such as one or more nucleotides (e.g., dNTPs, ddNTPs or derivatives thereof) or one or more polypeptides (e.g., enzymes) having reverse transcriptase activity and/or polymerase activity (e.g. DNA polymerases and or reverse transcriptases). Such nucleotides or derivatives thereof may include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, -thio-dATP, α-thio-dTTP, α-thio-dGTP, α-thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, ddGTP, ddlTP, 7-deaza-ddGTP, α-thio- ddATP, α-thio-ddTTP, -thio-ddGTP, α-thio-ddCTP or derivatives thereof, all of which are available commercially from sources including Life Technologies, Inc. (Rockville, Maryland), New England BioLabs (Beverly, Massachusetts) and Sigma Chemical Company (St. Louis, Missouri). Kits according to the invention may also comprise one or more enzymes such as endonucleases or restriction enzymes used for manipulating the vector of interest, and one or more cells competent for transformation (e.g. competent cells such as E. coli and the like). The kits encompassed by this aspect of the present invention may further comprise additional reagents (e.g., suitable buffers) and compounds necessary for carrying out the methods of the invention.

Examples Plasmid DNA Isolation Through Triple Helix Formation.

Introduction The triple helical structure of nucleic acids can be applied to isolate specific target genes from bulk DNA. A single-stranded homopyrimidine sequence can bind to the major groove of a double-stranded homopurine- homopyrimidine helix, through Hoogsteen hydrogen bonding. Several primidine-rich sequences designed from the replication origin and fl intergenic region sequences of the cloning vectors, such as pSPORTl, pCMVSPORT, BlueScript and the like have shown the capability of binding to the regions of corresponding sequences of vectors through the triplex structure. The biotinylated replication origin or fl origin-specific oligonucleotides have been used to isolate plasmid DNA from the crude DNA preparations.

Methods

The following sections describe the design of the vector specific oligonucleotides and specific isolation of the plasmid DNA.

Design of Oligonucleotides

Oligo Trip-1, CTTCCCTCTTTCCICCTITCC (SEQ ID NO: 45) was designed from the replication origin, which is parallel to the strand of GAAGGGAGAAAGGCGGACAGG (SEQ ID NO: 46). Oligo Trip-2, CTCTTTCCTTCCCTTCTTTC (SEQ ID NO: 47) was designed from the fl intergenic region, which is parallel to the strand of GAGAAAGGAAGGGAAGAAAG (SEQ ID NO: 48). A longer oligo Trip-3, CCICTCTTTCCTTCCCTTCTTTCICTTTCCTCICCC (SEQ ID NO: 49) was designed from the same fl intergenic region.

Example 1 Isolation of the plasmid DNA from Solution

Incubate 2 μg of leukocyte cDNA library and 2 μg of yeast tRNA with lOpmole of 3 '-biotinylated oligo (Trip-1, Trip-2 and Trip-3) in 100 μl of buffer 1 (2 M NaCl, 0.1 M sodium phosphate buffer, pH 6.0) at 50°C for 2 hours. After the 2 hour incubation, centrifuge the mixture for one second and sit at room temperature. Aliquot 100 μl of streptavidin-coated magnetic beads into centrifuge tube for each reaction. Wash the beads with 100 μl of Buffer 1. After removal of the buffer solution, immediately mix with the triplex reaction mixture. Incubate and mix the suspension for 1 hour at room temperature. After 1 hour incubation, insert the tubes into the magnet and allow them to sit for 2 minutes. Remove the supernatant and remove tube from magnet. Add 100 μl of Buffer 1, mix well, insert the tube in magnet for 2 minutes and remove the solution. Repeat the wash step two more times. Elute the captured DNA by adding 20 μl of Buffer 2 ( 1 M Tris, pH 9.0, 0.5 mM EDTA) to each reaction and incubating for 20 minutes at room temperature. Insert the tube into the magnet and wait for 2 minutes. Transfer the solution to a fresh tube, repeat the elution one more time, and pool the eluted solutions. Precipitate the DNA by adding 4 μl of 3 M NaAcetate and 100 μl of ethanol. The DNA pellet was dissolved in 30 μl of TE. 10 μl each was analyzed by gel electrophoresis. The results were shown in Figure 1.

Example 2 Selectivity of vector DNA Isolation

This experiment was designed to determine the purity of vector DNA isolated by the procedure. Vector DNA containing the targeted sequence fl intergenic region, pSPORT-CAT, was mixed with a 10-fold molar excess of a vector lacking this sequence, pSU41. Using bacterial transformation as a very sensitive assay, one can determine the presence of the second plasmid (kanamycin resistant) in a solution of the first (chloramphenicol resistant) by plating on media containing kanamycin. It is shown that the procedure differentially isolates only the vector containing the targeted sequence. Various mixtures of the vectors pSPORT-CAT (1.3 μg) and pSU41 (4 μg), with or without Trip-3 oligonucleotide (10 pmol), were prepared, then each except sample #9 was subjected to the purification protocol described in Example 1. The recovered DNAs were suspended in TE buffer and an aliquot was used to transform competent E. coli DH10B obtained from LTI according to the manufacture's protocols. Transformation reactions were plated on antibiotic selection plates containing either chloramphenicol (samples 1, 2, 5, 6, 7, and 8) or kanamycin (samples 3, 4, 8, and 9). The number of transformants on each plate per microliter of reaction are listed in the following table. These results clearly show that vector isolation by triplex formation using the fl intergenic region is highly specific and free of DNA contaminants.

Example 3 Isolation of Vector DNA from Partially-purified Cell Ly ates

This experiment was designed to evaluate whether vector DNA could be isolated from an impure cell extract using the procedure based on forming a vector/oligonucleotide complex. Briefly, vector DNA, pSPORT-CAT, was prepared from 2 ml of an overnight bacterial culture using either a modified alkaline lysis protocol [Birnboim, H. and Doly, J. (1979) Nucleic Acids Res. 7, 1513] or a lysozyme/boiling lysis protocol [Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) in Molecular Clomng: A Laboratory Manual, 2^nd ed., p.129].

In the case of alkaline lysis, vector DNA in the supernatant after centrifugation was further purified by either anion exchange chromatography using a commercial kit (Concert High Purity Plasmid Miniprep System, Life Technologies) or by the vector/oligonucleotide complex method as outlined in Example 1. Specifically for the latter, 1.2 ml of cleared lysate (pH 5-6) was mixed with -200 ng of Trip-3 oligonucleotide and incubated for 30 minutes at room temperature. Beads (100 μl) washed in Buffer 1 were added to the DNA mixture and incubated for 20 minutes at room temperature. The beads were washed once with 100 μl Buffer 1, then vector DNA was eluted from the complex by adding 30 μl Buffer 2, followed by a second elution with TE buffer.

In the case of lysozyme/boiling lysis, vector DNA in the supernatant after centrifugation was further purified by either alcohol precipitation or by the vector/oligonucleotide complex method as outlined in Example 1. Specifically for the latter, 415 μl of the supernant was mixed with 40 μl 2.5 M sodium acetate (pH 5.2) and -200 ng of Trip-3 oligonucleotide, then incubated for 30 minutes at room temperature. Beads (100 μl) washed in Buffer 1 were added to the DNA mixture and incubated for 20 minutes at room temperature. The beads were washed once with 100 μl Buffer 1, then vector DNA was eluted from the complex by adding 30 μl Buffer 2, followed by a second elution with TE buffer.

Samples of vector DNA purified by the four methods were analyzed by agarose gel electrophoresis (Figure 2). Vector DNA prepared using Trip-3 oligonucleotide complex to the specific target sequence, fl intergenic region of pSPORT-CAT, was quite pure, free of contaminating RNA as seen from the lysozyme/boiling lysis and free of chromosomal DNA seen from the alkaline lysis. The yield compared to the traditional methods were somewhat low.

Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Claims

Whatls Claimed Is:

1. A nucleic acid complex comprising one or more single stranded oligonucleotides and one or more single or double stranded vectors, wherein said oligonucleotide is bound to all or a portion of a functional sequence of said vector.

2. The complex of claim 1, wherein said functional sequence is selected from the group consisting of a gene, a translation sequence, a transcriptional sequence, a replication sequence and a tag sequence.

3. The complex of claim 1, wherein said functional sequence is selected from the group consisting of an antibiotic resistance gene sequence, a multiple cloning site sequence, a promoter sequence, a reporter or marker gene sequence, a recombination sequence, and an origin of replication sequence.

4. The complex of claim 1, wherein said oligonucleotides are bound to a support.

5. The complex of claim 4, wherein said support is a particulate support or bead.

6. The complex of claim 1, wherein said vectors are double stranded DNA vectors.

7. The complex of claim 1, wherein said oligonucleotide is a haptenylated oligonucleotide.

8. A method of separating the complex of claim 7 from a solution or sample that contains said complex comprising contacting said solution or sample with one or more ligand-coupled supports under sufficient conditions to bind said ligand-coupled supports to said haptenylated oligonucleotides and separating said complex from said solution or sample.

9. The method of claim 8, further comprising separating the complex from said supports.

10. The method of claim 8, further comprising separating the vectors from said oligonucleotides.

11. A kit for isolating one or more vectors from a solution or sample comprising the haptenylated oligonucleotides of claim 7 and one or more ligand-coupled supports.

12. The kit of claim 11, wherein said kit further comprises one or more solutions for removing contaminants, and/or for removing said one or more vectors from said support, and/or for removing said oligonucleotide from said one or more vectors.

13. A method for isolating or purifying one or more vectors from a sample comprising: contacting the sample with one or more support coupled oligonucleotides which are capable of binding to all or a portion of a functional sequence of one or more single or double stranded vectors under conditions sufficient to bind said oligonucleotides to said vectors thereby forming one or more vector-oligonucleotide complexes; and isolating said vectors from said one or more supports.

14. The method of claim 13, wherein said isolation is accomplished by separating or dissociating said one or more oligonucleotides from said one or more supports.

15. The method of claim 13, wherein said isolation is accomplished by separating or dissociating said one or more oligonucleotides from said one or more vectors.

16. The method of claim 13, wherein said oligonucleotides are coupled to said support by a ligand-hapten interaction.

17. The method of claim 13, wherein said oligonucleotides are bound directly to said support.