JP2002535987A - Isolation of nucleic acid molecules - Google Patents

Abstract

  (57) [Summary] The present invention relates to methods and kits for isolating or purifying single- or double-stranded vectors. Such isolated / purified vectors may interact with all or a portion of the functional sequences of the vector of interest and one or more oligonucleotides that target and bind one or more functional sequences. Obtained through. The resulting isolated / purified vector can be further manipulated or processed by well-known molecular biology techniques such as sequencing, digestion, ligation, transformation, amplification and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the purification and / or isolation of nucleic acids. The invention also relates to such isolated or purified nucleic acid molecules (eg, DNA and RNA) by well-known molecular biological techniques such as sequencing, digestion, amplification, transformation and / or synthesis.
) Further processing. In particular, the invention relates to the 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 (Felsenfe).
ld, G et al. (1957) J. Am. Am. Chem. Soc. 79: 2023-202
4). Although the biological role of such structures remains to be elucidated, their chemical characteristics have been largely elucidated in recent studies (for review, see Well
, R.A. D. (1988) FASEB J. et al. 2: 2939-2949). The best characterized triplex is that formed between the double-stranded homopurine-homopyrimidine helix and the single-stranded homopyrimidine region. In this type of triple helix, the third homopyrimidine chain is linked via a Hoogsteen hydrogen bond to a thick groove (parallel to the homopurine chain of Watson-Crick double helix DNA).
major groove). This third chain thymine (T) recognizes the adenine-thymine (AT) base pair and forms a TAT triplet. And the third chain cysteine (C) (protonated at its N-3 position) is guanine-
Recognizes cytosine (GC) base pairs and forms CGC triplets.

[0003] Typically, certain DNAs are synthesized using conventional hybridization-based methods (eg, colony or plaque hybridization (Sambrook,
J. et al., (1989) Molecular Cloning, A Laboratory.
ory Manual, 2nd edition, Cold Spring Harbor, Co.
ld Spring Harbor Press). These well-established methods are quite reliable, but have some practical disadvantages. First, they are time consuming and require a labor intensive step of filter preparation, which often limits the number of colonies that can be screened. In addition, these procedures include pre-denaturation steps and other treatments that destroy the integrity of the target DNA molecule. To obtain an intact DNA molecule for further biological and biochemical manipulations, the corresponding clone must be re-isolated from the original plate. Third, sequences that are toxic to the host sometimes prevent successful cloning. Fourth, natural modifications of the target DNA
Not maintained during cloning. Eventually, yeast artificial chromosomes (Yeast Arti)
physical Chromosome (YAC) vector and Bacterial Artificial Chromosome (B
AC) Despite the recent development of vectors, it is still difficult to clone very large DNAs. Clearly, non-cloning based biochemical methods for isolating specific DNA from a complex mixture will help some of these problems. However, such methods are not yet satisfactory. Biochemical purification by density and size fractionation after restriction enzyme digestion can be applied only in limited cases (Tsujimoto, Y, and Suzuki, Y (198).
4) Proc. Natl. Acad. Sci. USA 81: 1644-164.
8). The polymerase chain reaction (PCR) meets some of these needs and provides large amounts of DNA (Mullis, KB and Falocn).
a, F. A. (1987) Methods Enzymol 155: 335.
350), but it is typically limited to relatively short (<10 kb) DNA fragments. In addition, natural modifications of the original DNA cannot be maintained. DNA
An effective method of affinity chromatography has been reported for (Tsurui, H et al. (1990) Gene 88: 233-239), but it requires prior denaturation and elution by denaturation of the target DNA molecule.

[0004] Several screening procedures (potentially compatible with large DNA) that keep the target DNA in its native double-stranded form have been developed using the RecA protein (Honigberg, S. et al. 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). Affinity capture procedures using hybridization at the ends of large DNA fragments have also been reported (Kandpal, R et al. (1990) Nucleic Acids Res. 18: 1789-1795).
). However, these procedures, at least in some steps, involve the handling of DNA in solution, which necessarily breaks large DNA into smaller pieces.

[0005] Cantor et al. (US Pat. No. 5,482,836) and Wang et al. (US Pat. No. 5,401,632) have developed methods for capturing double-stranded plasmids using triple helix affinity. However, these methods rely on specialized plasmids that have been engineered to contain a target sequence to allow triple helix formation.

SUMMARY OF THE INVENTION [0006] The present invention provides a method for forming a complex between a target oligonucleotide and a target nucleic acid and separating the complex from the sample, preferably a biological sample. Improved methods for the isolation and / or purification of nucleic acid molecules from E. coli. The target nucleic acid molecule can then be recovered from the complex.
Unlike previous methods, the present invention provides nucleic acid molecules (preferably double- or single-stranded vectors, and most preferably double-stranded or single-stranded vectors) without the need to add or incorporate a target sequence into the nucleic acid molecule of interest. (Stranded DNA vectors). Thus, the present invention allows for the isolation / purification of a wide range of classes or groups of molecules or vectors commonly used in the field of molecular biology without having to construct specialized molecules or vectors.

[0007] Accordingly, the present invention provides a method for isolating and / or purifying a single-stranded or double-stranded vector (preferably a DNA vector) in a sample.
In a preferred aspect, the separation of the vector from the sample depends on the support, preferably a solid or semi-solid support. According to the invention, a sample is contacted with one or more oligonucleotides specific for one or more target vectors. Preferably, the oligonucleotide is linked to one or more haptens, either directly or indirectly. The haptenylated oligonucleotide-sample mixture is incubated under conditions sufficient to allow the oligonucleotide to bind to 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 attached. The interaction between the hapten of the oligonucleotide and the ligand of the support is determined by this vector-
Allow the oligonucleotide complex to be bound to the support. In another aspect, the oligonucleotide can be directly attached to a support, thereby avoiding the need for using a ligand / hapten interaction. The support bearing the complex is then separated from contaminants in the reaction mixture / sample, for example, by washing the support with a suitable solution (eg, buffer, water, etc.). The isolated / purified vector can then be obtained by removing the vector from the support. Preferably, the vector is separated from the support by treating the mixture with a reagent. This reagent disrupts or dissociates the interaction between the oligonucleotide and the vector without adversely affecting the single-stranded or double-stranded vector to be isolated and / or purified. Alternatively, the vector can be removed from the support by disrupting or dissociating the interaction between the hapten of the oligonucleotide and the ligand of the support. Then, the specific vector of interest is recovered. In a preferred aspect, the present invention provides a genomic library or cDN
It can be used to isolate / purify vector populations such as the A library.

[0008] In another aspect of the invention, the invention provides a purified and / or isolated vector that can be further manipulated by standard recombinant DNA techniques. Such techniques include, but are not limited to, amplification, transformation, digestion, sequencing, and the like.

[0009] In certain embodiments of the invention, one or more oligonucleotides are specific for a target sequence (or portion thereof) that is common to vectors. Such target sequences are typically functional sequences that impart useful phenotypic or genotypic characteristics to the vector or host cells containing the vector. Thus, the present invention is directed to such a common functional sequence, since the target sequence (or portion thereof) of the vector is a functional sequence that is typically found in many groups or classes of vectors. A general method is provided for isolating / purifying a large number of vectors having
Such sequences (or portions thereof) include an origin of replication, a promoter, a marker or selection gene (or sequence), an antibiotic resistance gene (or sequence),
Indicator gene (or sequence), repressor sequence, primer sequence, multiple cloning site sequence, termination sequence, transcription sequence, translation sequence, tag sequence, recombination sequence,
And portions thereof, but are not limited thereto.

[0010] According to the present invention, the hapten-ligand system for isolating / purifying the vector of interest can be any one or more haptens that bind to its corresponding one or more ligands. Such systems are readily recognized by those skilled in the art and include antigen / antibody, avidin / biotin, streptavidin / biotin, protein A / Ig and lectin / carbohydrate systems.

In accordance with the present invention, a support is any support capable of binding one or more ligands for isolating and / or purifying a vector of interest. Such a support may be a solid support or a semi-solid support, but is preferably a solid support. Supports for the present invention are plastic, glass, agarose,
Metal, nitrocellulose, acrylamide, silica, nylon (registered trademark), cellulose, diazocellulose, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, polydinyl fluoride (polydiriri)
nyl fluoride, sepharose, polyacrylamide, polystyrene divinylbenzene, polyvinyl toluene, modified polystyrene, polysaccharides, acrylic polymers, hydroxyapatite, agar, starch, nylon and latex. The form of such supports can vary to beads, particles, filters, columns, microtiter plates, and the like. Further, the support of the present invention can be magnetic, paramagnetic, or superparamagnetic. In a preferred aspect, the support is a bead, preferably a magnetic, paramagnetic, or superparamagnetic bead.

[0012] The present invention also provides a kit for isolating and / or purifying a vector of interest. Such a kit comprises any one of the kits for performing the methods of the invention.
One or more components may be included. This component includes one or more oligonucleotides of the present invention, one or more supports of the present invention, one or more solutions or buffers to remove contaminants (eg, washing solutions), and support materials. Contains one or more solutions for removing the vector of interest. Such kits may also include additional components for further manipulating or processing the isolated / purified vector.

[0013] The present invention also relates to a composition comprising a triple stranded complex comprising the single stranded oligonucleotide of the invention and a double stranded vector. The present invention also relates to a composition comprising a double-stranded complex comprising the single-stranded oligonucleotide of the present invention and a single-stranded vector. 2
Where more than one oligonucleotide is used to target the same target in the vector of interest, the composition of the invention may comprise a triple stranded complex (eg, two oligonucleotides and a single stranded vector), Chain complexes (eg, two oligonucleotides and a double-stranded vector) and the like. In a preferred aspect, the oligonucleotide can be linked (directly or indirectly) to one or more haptens or (directly or indirectly) to one or more supports. This composition also comprises
The support can include one or more ligands that can bind to the haptenized oligonucleotide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following description, a number of terms used in the fields of molecular biology and recombinant DNA technology are used extensively. The following definitions are provided to provide a clearer and more consistent understanding of the detailed description and claims, including the scope in which such terms are given.

Amplification As used herein, “amplification” refers to any in vitro method for increasing the number of copies of a nucleotide sequence through the use of a polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a nucleic acid (eg, DNA) molecule or primer, thereby forming a new nucleic acid molecule that is complementary to the nucleic acid template. The formed nucleic acid molecule and its template can be used as a template to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of multiple rounds of nucleic acid synthesis. The amplification reaction
For example, it includes the polymerase chain reaction (PCR). One PCR reaction may consist of 5-100 "cycles" of denaturing and synthesizing nucleic acid molecules.

(Gene) A DNA sequence containing information necessary for expression of a polypeptide or protein. It contains promoters and structural genes, as well as other sequences involved in the expression of the protein.

(Binding) As used herein, the terms “bind,” “bound,” or other similarly used terms refer to both covalent and non-covalent bonds, and / or interactions, Other molecular bonds such as Hoogsteen and Watson-Crick hydrogen bonds may also be included.

(Hybridization) The terms “hybridization” and “hybridize” refer to two complementary single-stranded nucleic acid molecules (RNA and / or DN).
A) refers to the pair that results in the double-stranded molecule of A). As used herein, two nucleic acid molecules can hybridize, but the base pairs are not completely complementary. Thus, incompatible bases may be used if appropriate conditions (known in the art) are used.
Does not prevent hybridization of two nucleic acid molecules.

(Host) Any prokaryotic or eukaryotic cell that is the recipient of the vector. The terms "host" or "host cell" may be used interchangeably herein. Examples of such hosts include Maniatis et al., Molecular.
Cloning: A Laboratory Manual, Cold Sp
ring Harbor Laboratory, Cold Spring H
arbor, New York (1982). Preferred prokaryotic hosts include, but are not limited to: Escherichi
a (eg, E. coli), Bacillus, Staphylococ
genus, Agrobacter (eg, A. tumefaciens),
Streptomyces, Pseudomonas, Salmonell
Bacteria such as genus a, Serratia, and Caryophanon. Most preferred prokaryotic hosts are E. coli. E. coli. Specific bacterial hosts of the present invention include E. coli. 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 (eg, Drosophila cells, Spodoptera).
Sf9 and Sf21 cells and Trichoplusa High-F
nematode cells (eg, C. elegans cells); and mammalian cells (eg, COS cells, CHO cells, VERO cells, 293 cells, PER)
C6 cells, BHK cells and human cells).

(Nucleotide) As used herein, “nucleotide” refers to a base-
Refers to a sugar-phosphate combination. Nucleotides are the monomer units (DNA and RNA) of a nucleic acid sequence. The term nucleotide refers to ribonucleoside triphosphates (eg, ATP, CTP, UTP, GTP), deoxyribonucleoside triphosphates (eg, 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 triphosphate (ddNTP) and derivatives thereof. Representative examples of dideoxyribonucleoside triphosphates include ddATP, ddCTP, ddGTP, ddIT
But not limited to P and ddTTP. According to the present invention,
A "nucleotide" may be unlabeled or detectably labeled by well-known techniques. Detectable labels include, for example, radioisotopes, 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, but may be a derivative of an oligonucleotide (eg, a peptide nucleic acid (PNA) oligonucleotide, a morpholino oligonucleotide, Pyrroleimidazole polyamide, etc.) are contemplated by this term.

(Vector) A vector is a single-stranded or double-stranded nucleic acid molecule (preferably DNA, preferably double-stranded and / or preferably circular) capable of autonomous replication in a host cell. Such vectors can also be characterized by having a small number of endonuclease restriction sites. At this site, such a sequence
The nucleic acid molecule can be cleaved without loss of essential biological function, and where the nucleic acid molecule can be spliced to achieve its replication and cloning. Therefore,
This vector may or may not contain the sequence of the gene of interest or other sequences (eg, a recombinant vector). Examples include plasmids, autonomously replicating sequences (ARS), centromeres, and phagemids. The vector may further provide primer sites (eg, for PCR), transcription and / or translation initiation and / or regulatory sites, recombination signals or sites, replicons, and the like. The vector may further include one or more selectable markers suitable for use in identifying cells transformed or transfected with the vector (eg, kanamycin, tetracycline, ampicillin, etc.).

According to the present invention, any vector can be used. In particular, vectors known in the art and commercially available vectors (and variants or derivatives thereof) can be used according to the present invention. Such vectors are, for example, Vector La.
boratories Inc. , InVitrogen, Promega, N
ovagen, NEB, Clontech, Boehringer Mannh
eim, Amersham Pharmacia Biotech, EpiCe
inter, OriGenes Technologies Inc. , Stra
tagene, Perkin Elmer, Pharmingen, Life
Technologies, Inc. , And Research Geneti
cs. Such a vector can then be used, for example, to clone or sub-clone a nucleic acid molecule of interest. General classes of vectors for specific purposes include prokaryotic and / or eukaryotic cloning vectors, expression vectors, recombinant cloning vectors, 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 (eg, PAC, YA
C and BAC).

Other vectors of interest include viral origin vectors (M13 and f1 vectors, bacterial phage λ vectors, baculovirus vectors, adenovirus vectors and retrovirus vectors), high copy number, low copy number and adjustable copy Number vectors, vectors with compatible replicons for use in combination in a single host (pACYC184 and pBR322), and eukaryotic episomal replication vectors (pCDM8).

[0025] Particular vectors of interest include prokaryotic expression vectors such as: pcDNAII, pSL301, pSE280, pSE380, pSE4.
20, pTrcHisA, pTrcHisB and pTrcHisC, pRES
ET A, pRESET B and pRESET C (Invitrogen,
Inc. ), PGEMEX-1 and pGEMEX-2 (Promega, In
c. ), PET vector (Novagen, Inc.), pTrc99A, pK
K223-3, pGEX vector, pEZZ18, pRIT2T, and pMC
1871 (Amersham Pharmacia Biotech, Inc.
), PKK233-2 and pKK388-1 (Clontech, Inc.).
, And pProEx-HT (LifeTechnologies, Inc.)
And variants and derivatives thereof. The vector can also be a eukaryotic expression vector such as: pFastBac, pFastBac HT, pF
astBac DUAL, pSFV and pTet-Splice (Life
Technologies, Inc. ), PEUK-C1, pPUR, pMAM
, PMAMneo, pBI101, pBI121, pDR2, pCMVENBNA
And pYACneo (Clontech), pSVK3, pSVL, pMSG
, PCH110, and pKK232-8 (Amersham Pharmac)
ia Biotech, Inc. ), P3'SS, pXT1, pSG5, pPb
ac, pMbac, pMC1neo and pOG44 (Stratagene,
Inc. ), And pYES2, pAC360, pBlueBacHis A
PBlueBacHis B and pBlueBacHis C, pVL13
92, pBuseBacIII, pCDM8, pcDNA1, pZeoSV, p
cDNA3, pREP4, pCEP4 and pEBVHis (Invitro
gen, Inc. ), As well as variants or derivatives thereof.

Other vectors of particular interest include: pUC18, pUC
19. pBlueScript, pSPORT, cosmid, phagemid, fosmid (pFOS1), YAC (yeast artificial chromosome), BAC (
Bacterial artificial chromosome), pBAC108L, pBAe3.6, pBeloBAC1
1 (Research Genetics), PAC, P1 (E. coli phage), pQE70, pQE60, pQE9 (Qiagen), pBS vector, ChargeScript vector, BlueScript vector, pNH
8A, pNH16A, pNH18A, pNH46A (Stratagene),
pcDNA3 (InVitrogen), pGEX, pTrsfus, pTrc
99A, pET-5, pET-9, pKK223-3, pKK233-3, pD
R540, pRIT5 (Amersham Pharmacia Biotec)
h), pSPORT1, pSPORT2, pCMVSPORT2.0, pSV-
SPORT1 (Life Technologies, Inc.), and provisional patent application No. 60 / 065,930 (filed October 24, 1997) and WO
99/21977, the entire contents of which are incorporated herein by reference.
And the variants or derivatives thereof.

[0027] Two-hybrid and reverse two-hybrid vectors of particular interest include: pPC86, pDBBlue, pDBTrp, pPC
97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pG
ADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGB
T9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pH
ISi-1, placZi, pB42AD, pDG202, pJK202, pJ
G4-5, pNLexA, pYESTrp, and variants and derivatives thereof.

Examples of target sequences contained in a vector that can be used in the isolation / purification procedures of the present invention include any sequences or portions thereof commonly found in vectors. Such sequences are those that have not been used to isolate / purify the vector. Preferably, such sequences are conferring phenotypic and / or genotypic properties on the vector or on the host containing the vector, or on the product or protein (peptide) encoded by the vector. It is a functional array. For example, target sequences include, but are not limited to, any gene or portion thereof linked to a vector such as: an antibiotic resistance gene or portion thereof (eg, tetracycline, ampicillin, neomycin, kanamycin) , Chloramphenicol, etc.), a reporter gene or marker gene or a part thereof (eg, β-galactosidase gene, luciferase gene, lacZ, green fluorescent protein, chloramphenicol transferase), a suicide gene and / or a death gene or a part thereof (Eg, ccdB lethal gene, Dpn1 methylase, sucB (sucrose sensitivity, streptomycin L and restriction enzyme genes (eg, Bam HI
, Eco RI, Hae III, etc.), any gene or part thereof encoding a particular protein / peptide of interest, any termination sequence or part thereof, any primer sequence or part thereof (eg, standard sequencing M13) Forward primer or M13 reverse primer), any multiple cloning site sequence or part thereof, any translation functional sequence or part thereof (eg, cap structure (
m ⁷ GpppN), Shine-Dalgarno (SD) sequences, poly (A) and poly (dA) tails), any transcriptional functional sequences or parts thereof (eg, promoter, polyadenylation signal, splice site, enhancer sequence), Any replication sequence or portion thereof (eg, plasmid origin of replication, viral origin of replication, eukaryotic origin of replication, F1 intergenic region), any protein tag sequence or portion thereof (eg, GST, His, Tag, MBP, ZZ) , Pinpoint (pinp
oint), strep tag, Flag peptide), and any recombinant sequences or portions thereof (eg, attB, attL, attP, attR, loxP).
), (And variants or derivatives thereof) and PCT application WO 96/40
724. The target sequence according to 724.

Since the sequences of many vectors are known, a person skilled in the art can easily select an appropriate target sequence for the oligonucleotide design according to the present invention. Alternatively, the vector can be easily sequenced by standard techniques, so that the appropriate target can be selected. The conditions for forming the oligonucleotide-nucleic acid target complex, and the length of the oligonucleotide, can vary depending on the target sequence used. Such conditions, as well as the type and length of the oligonucleotide, can be determined by standard binding assays (eg, transformation assays or gel assays as described in the Examples).
Can be determined and optimized. To isolate a double-stranded vector target, the purine-pyrimidine-rich target sequence is linked, preferably using a pyrimidine-rich oligonucleotide, thereby forming a triple-stranded molecule. For sequences consisting of purine nucleotides, nucleotide derivatives (eg, deoxyinosine (I), 9- (1-β-D-2-deoxyribofuranosyl) -2-
An oligonucleotide of the invention is constructed using amino-6-methoxyaminopurine (dK) and / or 3-nitropyrrole deoxyribonucleoside (M or dM), and the oligonucleotide is converted to a double-stranded vector. It may allow for efficient coupling. Other such derivatives for use in the present invention will be readily apparent to those skilled in the art. Accordingly, the invention relates to such derivative oligonucleotides used in the method of the invention.

A list of base analogs that can replace the bases adenine and guanine in the capture sequence includes, but is not limited to:

[0031]

Embedded image Table 1 shows a number of oligonucleotide sequences specific for a particular vector target region, including the origin of replication, intergenic regions and gene sequences.

[0032]

[Table 1]

[0033]

[Table 2] Other target sequences normally found in vectors include the F1 intergenic region,
Origin of replication, lacI, lacZ, lacA, lacY, antibiotic resistance gene, luciferase gene, and SV40 intron and polyadenylation signal. In accordance with the present invention, one or more oligonucleotides can be prepared to target such sequences or portions thereof.

Examples of vectors containing the F1 intergenic region include the following: yeast integration vector, cloning vector pALTER-1, cloning vector pGEM-5Zf (+), pBluescript II SK (+) vector DNA, pBluescript II. KS (+) vector DNA, yeast episomal vector pRS426, yeast integration vector pRS305, pICEM
19R minus plasmid cloning vector, cloning vector pGE
M-13Zf (+), plasmid pKA1 DNA, yeast centromere vector pRS414, yeast centromere vector pRS413, pEMBL 9 minus plasmid cloning vector, pEMBL 8 minus plasmid cloning vector, yeast centromere vector pRS415, cloning vector pEMBL 8 minus (pEMBL8m), cloning Vector pGEM
-7Zf (+), yeast centromere vector pRS416, pBluescript
pt SK (+) vector DNA, yeast centromere vector pRS314, p
Bluescript KS (+) vector DNA, BlueScript K
S + cloning vectors and the like, as well as variants or derivatives thereof.

Examples of vectors containing the replication origin sequence include the following: cloning vector pLXSH, cloning vector pGEMEX-2, pSP6T71
9 cloning vector, pJSC73 cloning vector (derived from pBR325 and pAT153), multicopy Saccharomyces cere
visiae / E. coli shuttle vector, YEp24 yeast extrachromosomal plasmid, pMC1511 cloning vector, pEx3 expression vector, plasmid pKUN, cloning vector, synthetic cloning vector plasmid pH
SG664, cloning vector cosmid pTCF, yeast episomal vector pRS425, cloning vector pDR2, pFNeo eukaryotic expression vector; coli plasmid synthesis cloning vector pET31F1P, yeast centromere vector pRS315, and the like, and modified or derivatives thereof.

Examples of vectors containing the lacI, lacZ, lacY and lacA genes include: the cloning vector pFRT2, M13mp11,
Phage cloning vector, M13mp10 phage cloning vector, integration vector pMUTIN2, pUR290 cloning vector, cloning vector pNASSbeta, cloning vector M13mp18,
M13mp9 phage cloning vector, cloning vector pPD21
. 28, expression vector pUEX2, pGEX-6P-3 cloning vector,
Plasmid pLGlacz7, pCH110 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, etc., and modified or derivatives thereof.

Examples of vectors containing the antibiotic resistance gene include the following: shuttle vector pHY320PLK DNA, cloning vector pEG202 (
pLexA), cloning vector pGL2-promoter, cloning vector pGEM-4Z, BlueScribe cloning vector, cloning vector pKK388-1, plasmid pUT18, plasmid pH251
5, shuttle vector, reporter vector pCRE-Luc, cloning vector pJG4-5 (pB42AD), cloning vector pTRE, cloning vector pDG1731, ligation-independent cloning vector pBlue
script, 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 containing a luciferase gene include: a eukaryotic luciferase expression vector pCMVtkLUC +, a cloning vector pGL3-Promoter, a cloning vector pSP-luc + NF, a eukaryotic luciferase expression vector pLUC +, and a cloning vector pGL3.
-Enhancer, cloning vector pVLH-1, cloning vector pGL3-Basic, eukaryotic luciferase expression vector ptkLUC +
Cloning vector pGL3-Control, cloning vector pS
P-luc +, eukaryotic luciferase expression vector pTATALUC +, reporter vector p2luc, cloning vector pFRLuc, expression vector pBSII-LUCIN, expression vector pZE1PA1lacO-1 luc,
Cloning vector pGL2-Basic, cloning vector pMMn
eo-LUC, cloning vector pGL2-Control, and variants or derivatives thereof.

Examples of vectors containing the SV40 intron and the polyadenylation signal include the following: cloning vector pFAC-dbd, signal sequence detection vector pSSD3, cloning vector pEGFP-N2, expression vector pNEX, cloning vector pMAMneoBlue, expression vector pVP.
HA1, cloning vector pSV2neo, cloning vector pCM
VTAG4a, epitope-tagged vector pCMV-Tag1, cloning vector pSEAP-Control, ligation-dependent promoter cloning vector, reporter vector pSRF-Luc, cloning vector pBI-
GL, cloning vector pMUT-Elk, cloning vector pFA
2-elkl, co-reporter vector pRL-SV40, expression vector pNEX
δ, cloning vector pCMVTAG5a, cloning vector pCM
V-scriptEX, reporter vector pCAT3-Enhancer vector, and variants or derivatives thereof.

(Isolation of Nucleic Acid Molecules) According to the present method, single-stranded 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 molecule to a support. According to the invention, a vector is isolated / purified through the binding of one or more oligonucleotides to one or more target sequences on the vector of interest. In this way, the nucleic acid molecule of interest can be isolated / purified from any sample or reaction mixture.

The vector can be an RNA or DNA vector, double or single stranded, circular or linear, or any combination thereof. If the vector is single stranded, the oligonucleotide is under conditions sufficient to bind or hybridize to the single stranded vector to form a double stranded molecule, wherein the oligonucleotide is Binds to this vector target sequence. If the vector is double-stranded, the oligonucleotide under appropriate conditions,
Binds to a double-stranded vector to form a triplex molecule, wherein the oligonucleotide binds to the vector target sequence. In another aspect, the plurality of oligonucleotides specific for a single target site bind to a single-stranded or double-stranded vector,
Thereby, a complex such as a triplex or a quadruplex can be formed. 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. With such multiple oligonucleotides / multiple target sequences, the same or different vectors can be isolated and used sequentially or simultaneously. Thus, the present invention allows for the isolation / purification of many different vectors from one sample using different targets. Alternatively, increasing the selectivity or purity of a single type of vector can be achieved using a number of different target sequences for a single vector of interest. In addition, multiple haptens can be used in combination with multiple types of oligonucleotides or a single type of oligonucleotide to allow for increased binding or higher binding capacity for binding of the oligonucleotide vector to the support. Similarly, multiple different ligands can be used on a support, or multiple supports, each with a different ligand, can be used in the present invention. Thus, the use of multiple supports may provide a means for isolating / purifying multiple vectors from one sample.

In the practice of the present invention, any support may be used to bind oligonucleotides or hapten-specific ligand molecules. Preferably, a solid support is used. Preferred such solid supports include, but are not limited to, nitrocellulose, diazocellulose, glass, silica, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads. And microtiter plates. Preferred are beads made of glass, latex or magnetic material, and particularly preferred are magnetic, paramagnetic or superparamagnetic beads. Attachment of the ligand molecule or oligonucleotide to the support may be by any method (eg, covalent, non-covalent, hydrophobic, or ionic coupling (including coating), as is conventional in the art). Can be achieved.

According to the present invention, any hapten molecule capable of binding to a ligand molecule can be used. Particularly preferred hapten / ligand molecules for use in the present invention include, but are not limited to: (i) avidin and streptavidin; (ii) protein A, protein G, cell surface Fc receptor or antibody specific antigen (Iii) enzyme-specific substrate; (iv) polymyxin B or endotoxin-neutralizing protein (ENP); (v) Fe; (vi) transferrin receptor; (vii) insulin receptor; (viii) cytokines (eg, growth factors, (Ix) CD4; (x) spectrin or fodrin; (xi) ICA
M-1 or ICAM-2; (xii) C3bi, fibrinogen (fibr)
(xiii) ankyrin; (xiv) integrin α ₁ β ₁ , α ₂ β ₁ , α ₃ β ₁ , α ₆ β ₁ , α ₇ β ₁ and α ₆ β ₅ ; (xv) integrin α ₃ β ₁ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α _v β ₁ , α _iib β ₃ , α _v β ₃ and α _v
β _6; (xvii) integrin alpha _v beta ₁ and α _v β _3; (xviii) vitronectin; (xix) fibronectin; (xx) collagen; (xxi) laminin; (xxii) glycophorin; (xxiii) Mac-1; ( xxiv) LF
(Xxv) β-actin; (xxvi) gp120; (xxvii) cytokine (growth factor, interleukin or colony stimulating factor); (xxv
iii) insulin; (xxix) ferrotransferrin; (xxx) apotransferrin; (xxxi) lipopolysaccharide (saccharide); (xxxiii) enzyme; (xxxiii) antibody; and (xxxiv) biotin. Hapten binding to the oligonucleotides of the invention can 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 (eg, avidin or streptavidin) can be linked to the oligonucleotide. Alternatively, the support ligand can be avidin or streptavidin, and the oligonucleotide hapten can be biotin. In particularly preferred such aspects, the solid support used is, for example, Dyn
al A. S. (Oslo, Norway), Seradyne (Indian
apolis, IN) or Sigma (St. Louis, Missouri).
) Are magnetic, paramagnetic or superparamagnetic beads bound to avidin or streptavidin. Of course, the choice of ligand will depend on the choice of hapten used; therefore, suitable ligands for use in the methods of the invention are routine to those of skill in the art.

To isolate a nucleic acid molecule produced by a method of the invention, a solution or sample containing the vector complex is contacted with one or more oligonucleotides under conditions that favor binding. The oligonucleotide can bind indirectly to one or more haptens that can be bound to one or more supports through ligand-hapten interactions.
In a preferred aspect, a host cell containing the vector of interest is lysed using well known techniques (eg, chemical, enzymatic or mechanical lysis), and the crude extract containing the vector is lysed using a haptenized oligonucleotide. And a ligand-bound support or an oligonucleotide-bound support. Alternatively, the reaction mixture in which the vector has been manipulated (eg, digestion, amplification, ligation, etc.) can be used as a sample for isolating / purifying the vector. Typically, conditions for contacting the vector with the oligonucleotide and the support include a buffered salt solution (preferably,
TRIS-, phosphate-, HEPES or carbonate-buffered sodium chloride solution, more preferably TRIS-buffered sodium chloride solution, even more preferably about 10-100 mM TRIS-HCl and about 300-2000 mM NaC.
l, and most preferably a solution containing about 0.1 M sodium phosphate buffer and about 2 M NaCl) in the presence of about 4-9, more preferably about 4-8.
PH, even more preferably at a pH of about 4.5 to 6.5, and most preferably at a pH of about 6.0. Incubations are preferably at 0 ° C to about 60 ° C, and most preferably at about 50 ° C for about 30-240 ° C.
Minutes, preferably for about 45 to 120 minutes, and most preferably for about 120 minutes.

Once one or more vectors are bound to a support, undesired or contaminated 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.) can be eliminated by simply removing them from the supernatant. For example, in a preferred aspect, in which the vector of interest is bound to beads or other particulate support, a mixture comprising the vector is mixed and under sufficient conditions and for a time and under conditions sufficient for the vector to bind to the bead or support. After incubation, the vector-bead complex is then separated from the mixture by any physical means (eg, filtration, centrifugation, magnetic means, etc.), and any remaining supernatant containing the contaminants is aspirated. , Pipetting and the like. The vector-support complex or vector-bead complex is then washed any number of times with any solution (preferably one or more buffers) that is compatible with the vector complex of interest to further remove contaminants. Can be removed. In a particularly preferred aspect, magnetic, paramagnetic or supermagnetic beads with attached ligand are used as a support, and the vector-bead complex is a magnet (eg, Magna-Sep Magnetic Par- tic).
life Separator; Life Technologies, In
c. ) To separate from the supernatant. Prior to their release from the support, the immobilized vector is preferably washed one or more times, for example with one of the buffered salt solutions described above, to reduce the undesired substances to a greater extent. Is removed.

Once the contaminants have been substantially or sufficiently removed, the vector molecule can be supported with any solution that can dissociate or remove the vector from the oligonucleotide and / or dissociate the hapten-ligand interaction. It can be released from the support by contact with the body. Preferred conditions for release of the vector from the support include elevated temperatures, changes in pH and / or changes in salt concentration. Preferably, such release is achieved by changing the pH. After their release from the support, the vectors can be prepared using techniques well known in the literature (eg, digestion, ligation, amplification, nucleic acid synthesis, transformation into one or more host cells, and sequencing).
(See, eg, Sambrook, J. et al., Molecular Cloning:
A Laboratory Manual, 2nd edition, Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory
ory Press, pages 8.60-8.63 (1987)) and other techniques routine to those skilled in the art. Particularly preferred amplification methods according to this aspect of the invention include PCR (US Pat. No. 4,683,683).
, 195 and 4,683,202), strand displacement amplification (Stranded).
Displacement Amplification: SDA; U.S. Patent No. 5,455,166; EP 0 684 315), and nucleic acid sequence based amplification (Nucleic Acid Sequence-Based Ampl).
application: NASBA; US Pat. No. 5,409,818; EP 0
329 822). Most preferred are methods that include one or more PCR amplifications.

(Kit) The present invention also provides a kit used for isolation and / or purification of a vector of interest and a kit for further manipulation or processing of the isolated / purified vector as required. . Kits according to this aspect of the invention include a carrier, such as a box, carton, tube, etc., which is sealed in one or more such as vials, tubes, ampules, bottles, microtiter plates, etc. Wherein the kit comprises one or more oligonucleotides of the invention, which are preferably haptenylated, one or more supports or ligand binding supports, And one or more components selected from the group consisting of one or more wash buffers to remove unwanted contaminants and one or more release buffers to remove vectors from the support. In a further aspect, the kit of the present invention comprises instructions for isolating / purifying a vector of interest and 1
One or more nucleotides (eg, dNTPs, ddNTPs or derivatives thereof), or reverse transcriptase and / or polymerase activities (eg,
It may further include other factors such as one or more polypeptides (eg, enzymes) having DNA polymerase and / or reverse transcriptase). Such nucleotides or derivatives thereof include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, α-thio-dATP, α-thio-dTTP, α. -Thio-dGTP,
α-thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, dd
GTP, ddITP, 7-deaza-ddGTP, α-thio-ddATP, α-thio-ddTTP, α-thio-ddGTP, α-thio-ddCTP or derivatives thereof (all of these are Life Technologies, Inc. (Roc.
kville, Maryland), New England BioLabs
(Beverly, Massachusetts) and Sigma Chem
ical Company (commercially available from sources including St. Louis, Missouri)). The kit according to the invention may also comprise one or more enzymes such as endonucleases or restriction enzymes used to manipulate the vector of interest.
As well as one or more cellular components for transformation (eg, component cells such as E. coli). This kit included by this aspect of the invention,
In addition, it may include additional reagents (eg, appropriate buffers) and compounds necessary to perform the methods of the invention.

EXAMPLES Isolation of Plasmid DNA Via Triple Helix Formation Introduction The triple helix structure of nucleic acids can be applied to the isolation of specific target genes from bulk DNA. Single-chain homopyrimidine
2.) The sequence can bind to the major groove of the double-stranded homopurine-homopyrimidine helix via a Hoogsteen hydrogen bond. Cloning vectors (eg, pSPOR
T1, pCMVSPORT, BlueScript, etc.) and f1
Several pyrimidine-rich sequences designed from intergenic region sequences have shown the possibility of binding the corresponding sequence of the vector to the region via a triple helix structure. The biotinylated origin of replication or f1 origin specific oligonucleotide comprises the crude DNA
It has been used to isolate plasmid DNA from preparations.

Methods The following sections describe the design of vector specific oligonucleotides and plasmid D
Describes the specific isolation of NA.

Oligonucleotide Design Oligo Trip-1, CTTCCCCTCTTCCCICCTITCC (SEQ ID NO: 45) was designed from an origin of replication, which is GAAGGGGAGAAAGGCG
This corresponds to the chain of GACAG (SEQ ID NO: 46). Oligo Trip-2, CTCT
TTCCTTCTCCTTCTTTC (SEQ ID NO: 47) was designed from the f1 intergenic region, which was GAGAAAGGGAAGGGAAGAAAG (SEQ ID NO: 48).
). Longer oligo Trip-3, CCICTCTTTCCTT
CCCTTCTTTCICTTTCCTCCICCC (SEQ ID NO: 49) has the same f
It was designed from one intergenic region.

Example 1 (Isolation of Plasmid DNA from Solution) 2 μg of leukocyte cDNA library and 2 μg of yeast tRNA were
mol of 3'-biotinylated oligo (Trip-1, Trip-2 and T
rip-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 a 2 hour incubation, the mixture is centrifuged for 1 second and placed at room temperature. Aliquot 100 μl of streptavidin-coated magnetic beads into centrifuge tubes for each reaction. The beads are washed with 100 μl of buffer 1. Immediately after removal of the buffer solution, mix with the triplicate reaction mixture. The suspension is incubated for 1 hour at room temperature and mixed. After a 1 hour incubation, insert the tubes into a magnet and place them for 2 minutes. Remove the supernatant and remove the tube from the magnet. Add 100 μl of Buffer 1, mix well, insert the tube into the magnet for 2 minutes and remove the solution. The washing step is repeated twice more. The captured DNA was added to each reaction with 20 μl of Buffer 2 (1
M Tris, pH 9.0, 0.5 mM EDTA) and elute by incubating for 20 minutes at room temperature. Insert the tube into the magnet and wait 2 minutes. Transfer the solution to a new tube, repeat the elution one more time, and pool the eluted solution. DNA is precipitated by adding 4 μl of 3M sodium acetate and 100 μl of ethanol. D
Dissolve the NA pellet in 30 μl TE. Each 10 μl was analyzed by gel electrophoresis. The results are shown in FIG.

Example 2 Selectivity of Vector DNA Isolation This experiment was designed to determine the purity of vector DNA isolated by this procedure. Vectors containing the f1 intergenic region of the targeted sequence (pSP
(ORT-CAT) in a vector (pSU) lacking a 10-fold molar excess of this sequence.
41). Bacterial transformation can be used as a very sensitive assay to determine the presence of a second plasmid (kanamycin resistance) in a first solution (chloramphenicol resistance) by plating on media containing kanamycin. . This indicates that only the vector containing the target sequence is separately isolated by this procedure.

The vector pSPORT-CAT (1.3 μg) and the vector pSU41 (4
μg) of the various mixtures are prepared with or without Trip-3 oligonucleotide (10 pmol), and each sample except sample # 9 is subjected to the purification protocol described in Example 1. The recovered DNA is suspended in TE buffer and aliquots are used to determine LT according to the manufacturer's protocol.
Competent E.I. coli DH10B. 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 per μl of reaction in each plate is listed in the table below. These results indicate that the vector isolated by triple formation using the f1 intergenic region is more specific and
It clearly shows no NA contamination.

[0055]

[Table 3] Example 3 Isolation of Vector DNA from Partially Purified Cell Lysate This experiment demonstrates that vector DNA was isolated from impure cell extracts using a procedure based on the formation of a vector / oligonucleotide complex. It was designed to evaluate whether it could be isolated. Briefly, vector DNA, pSPORT-CAT, was
Overnight modified bacterial lysis protocol [Bi
rnboim, H .; And Doly, J .; (1979) Nucleic Aci
ds Res. 7, 1513] or lysozyme / boiling lysis protocol [Sambrook, J. et al. Fritsch, E .; F. , And Maniati
s, T. (1989) Molecular Cloning: A Labora
toy Manual, 2nd edition, p. 129].

In the case of alkaline lysis, the vector DNA in the supernatant after centrifugation is converted to a commercially available kit (Concert High Purity Plasmid Miniprem).
Further purification was performed either by anion exchange chromatography using pSystem, Life Technologies or by the vector / oligonucleotide conjugate method outlined in Example 1. In detail for the latter, 1.2 ml of clarified lysate (pH 5-6) is added to ２００200 ng of T
Mix with rip-3 oligonucleotide and incubate 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 of buffer 1 and then the vector DNA was eluted from the complex by the addition of 30 μl of buffer 2 followed by a second elution with TE buffer.

In the case of lysozyme / boiling lysis, the vector DNA in the supernatant after centrifugation is further purified by either alcohol precipitation or the vector / oligonucleotide complex method as outlined in Example 1. did. In detail for the latter, 415 μl of the supernatant was mixed with 40 μl of 2.5 M sodium acetate (pH 5.2) and ２００200 ng of Trip-3 oligonucleotide, then incubated for 30 minutes at room temperature. The beads (100 μl) washed with buffer 1 were added to DN
A mixture was added and incubated for 20 minutes at room temperature. These beads
Washing once with 100 μl of buffer 1 then vector DNA was eluted from the complex by addition of 30 μl of buffer 2 followed by a second elution with TE buffer.

[0058] Samples of vector DNA purified by the four methods were analyzed by agarose gel electrophoresis (Figure 2). Vector DNA prepared with the Trip-3 oligonucleotide complex to a specific target sequence (p1PORT-CAT f1 intergenic region) is very pure and contains RNA contamination and as seen from lysozyme / boiling lysis. There was no chromosomal DNA seen from alkaline lysis. The yield was somewhat lower compared to the conventional method.

The invention has now been described in some detail and by way of illustration and example for clarity of understanding. The same does not alter or alter the invention, within the breadth and equivalents of the conditions, formulations and other parameters, without affecting the scope of the invention or any particular embodiment thereof. It will be apparent to those skilled in the art that modifications and variations may be made, and that such modifications or changes are intended to be included within the scope of the appended claims.

All publications, patents, and patent applications cited herein indicate the level of ordinary skill in the art to which the present invention pertains, and which are used herein to refer to each individual publication, patent, or patent application. It is incorporated by reference to the same extent as if the patent application was specifically and individually indicated to be incorporated by reference.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the isolation of plasmid DNA by formation of a triple-stranded complex. Lane M
1 kb DNA ladder; lanes 1 and 2: no oligo added; lanes 3 and 4: added oligo Trip1; lanes 5 and 6: added oligo Trip2; and lanes 7 and 8: added oligo Trip3.

FIG. 2 shows a comparison of plasmid DNA isolates from crude cell extracts. Lane M
1 kb DNA ladder; Lane 1: Concert Rapid Plas
DNA was isolated using the kit of the Mid Miniprep System;
Lane 2: Concert Rapid Plasmid Miniprep
DNA was isolated using the System kit, followed by purification of triplex formation; lane 3: DNA was isolated with the lysozyme / boiling lysis protocol;
And Lane 4: DNA was isolated by lysozyme / boiling lysis followed by purification of triplex formation.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, (72) Invention NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW Blakesley, Robert W .. United States Maryland 21702, Frederick, Stone Ridge Drive 8193 (72) Inventor Li, Uvo United States of America Maryland 20878, Gaithersburg, Riding Loop Terrace 11909 (72) Inventor Press, Reynolds Sea. Dee, Mexico. F. 11560, Mexico, Delegation Miguel Hidalugo, Colonia Polanco, 6 Oh. Piso, Brass Pascal 206 F term (reference) 4B024 AA20 CA04 CA11 HA14 HA19

Claims (17)

[Claims] 1. A nucleic acid complex comprising one or more single-stranded oligonucleotides and one or more single-stranded or double-stranded vectors, wherein the oligonucleotides comprise a functional sequence of the vector. A nucleic acid complex that binds to all or part of the nucleic acid complex. 2. The complex according to claim 1, wherein said functional sequence is selected from the group consisting of a gene, a translation sequence, a transcription sequence, a replication sequence, and a tag sequence. 3. The 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 a replication origin sequence. Item 1
The complex according to any one of the above. 4. The method according to claim 1, wherein the oligonucleotide is bound to a support.
The complex according to any one of the above. 5. The conjugate of claim 4, wherein said support is a particle support or beads. 6. The complex according to claim 1, wherein said vector is a double-stranded DNA vector. 7. The conjugate of claim 1, wherein said oligonucleotide is a haptenized oligonucleotide. 8. A method for separating a complex from a solution or a sample containing the complex according to claim 7, wherein the complex is separated under a condition sufficient to bind a ligand-bound support to the haptenized oligonucleotide. Contacting said one or more ligand binding supports with said solution or said sample; and separating said complex from said solution or said sample. 9. The method of claim 8, further comprising the step of separating said complex from said support. 10. The method of claim 8, further comprising the step of separating said vector from said oligonucleotide. 11. The haptenated oligonucleotide according to claim 7, and 1
A kit for isolating one or more vectors from a solution or sample containing one or more ligand binding supports. 12. The kit according to claim 11, wherein the kit is for removing contaminants and / or for removing the one or more vectors from the support, and / or A kit further comprising one or more solutions 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-bound oligonucleotides; The oligonucleotide can bind to all or a portion of the functional sequence of one or more single-stranded or double-stranded vectors under conditions sufficient to bind the oligonucleotide to the vector. Forming one or more vector-oligonucleotide conjugates; and isolating said vector from said one or more supports. 14. The method of claim 13, wherein said isolating 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 isolating 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 oligonucleotide is bound to said support by a ligand-hapten interaction. 17. The method according to claim 17, wherein the oligonucleotide is directly bound to the support.
The method according to claim 13.