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WO2006114834A1 - Procede d’elaboration d’un vecteur permettant d’introduire un groupe fonctionnel specifique a un point - Google Patents

Procede d’elaboration d’un vecteur permettant d’introduire un groupe fonctionnel specifique a un point Download PDF

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Publication number
WO2006114834A1
WO2006114834A1 PCT/JP2005/006830 JP2005006830W WO2006114834A1 WO 2006114834 A1 WO2006114834 A1 WO 2006114834A1 JP 2005006830 W JP2005006830 W JP 2005006830W WO 2006114834 A1 WO2006114834 A1 WO 2006114834A1
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Prior art keywords
primer
ligand
bound
dna
functional group
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Japanese (ja)
Inventor
Toshiya Arakawa
Toru Ota
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Hokkaido Technology Licensing Office Co Ltd
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Hokkaido Technology Licensing Office Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the present invention relates to a novel DNA functional group introduction method capable of introducing a functional group point-specifically into a DNA sequence.
  • the method of the present invention is useful as a technique for studying the effect on transcription by adding a functional group to a transcription region.
  • DNA methylation is the only chemical modification found in genomic DNA in mammals. Higher organism DNA consists of four types of bases, but contains 5-methylol nucleic acid bases as trace bases. Such DNA methylation is considered to have two main significance. The first is a self-protection system against DNA (parasite) that enters from the outside, and they are known to be thoroughly methylated and inactivated. This is because the transcription region is methylated and transcription factors cannot bind.
  • the second is regulation of self-gene expression.
  • the transcriptional activity of the gene is inactivated, thereby regulating the expression of the gene, that is, when the nucleobase in DNA is methylated, the gene expression Is known to be negatively controlled.
  • DNA binding protein It is collectively referred to as a DNA binding protein, and this DNA binding protein has a site that exhibits a strong affinity for the target DNA.
  • a DNA-binding protein and DNA can be detected by a protein-DNA conjugate.
  • a protein-DNA conjugate used for such detection one in which a protein is bound to a specific site of DNA is considered preferable.
  • the DN A-drug complex with a drug bound to a specific site of DN A can be applied as a drug delivery system.
  • various proposals have been made such as binding an amide group to DNA and binding a protein or the like to the amide bond. Although it has been made, it has not been known so far that it can efficiently form a complex at a specific site.
  • an object of the present invention is to provide a novel DNA functional group introduction method capable of introducing a functional group point-specifically into the DNA sequence. That is, an object of the present invention is to provide a DNA functional group introduction method useful as a technique for studying the effect on transcription by imparting a functional group to a transcription region.
  • the present invention has been made on the basis of the above knowledge, and provides a DNA functional group guide method comprising a step of performing a PCR method using the following primers (i) to (ii): .
  • a forward primer designed to increase a specific region of a long-form DNA, a repurse primer, and a ligand bound to the 5 'terminal side of the repurse primer In addition, it is designed to amplify a product that lacks the same number of bases as the base C of the forward primer 1 on the ligand-bound reverse primer and the product amplified by the forward primer 5.
  • a ligand-bound forward primer having a ligand bound to the 5′-end side, wherein the forward primer has a base sequence including a nucleobase bound to a functional group.
  • the number of bases equal to the number of bases of the forward primer and the 5 'end of the product amplified by the reverse primer. It is designed to amplify the lost product and contains a ligand-bound reverse primer with a ligand S binding at the 5 'end.
  • the reverse primer has a nucleotide sequence including a nucleobase bound to a functional group.
  • the second primer set, wherein the repurse primer has three bases complementary to the forward primer included in the first primer set.
  • the present invention also includes: (A) using the primers (i) and (ii) below: performing PCR; (i) a forward designed to amplify a specific region of a truncated DNA Primer and reverse primer, ligand-bound reverse primer in which a ligand is bound to the 5 ′ end of the reverse primer, and the 5 ′ end side of the product amplified by the forward primer are placed on the forward primer.
  • the first primer set which has a base sequence containing a nucleobase bound to (7) a primer sequence designed to amplify a specific region of mirror-type DNA A berth primer, a ligand-bound repurse primer with a ligand bound to the 5 ′ end of the repurse primer, and the number of salt tombs that the forward primer has on the 5 ′ end of the product amplified by the forward primer It is designed to amplify products lacking the same number of bases, and includes a ligand binding forward primer having a ligand bound to the 5 ′ end, and the forward primer is a functional group.
  • a method for introducing a D N A functional group is provided.
  • the present invention provides a method for constructing a point-specific functional group introduction vector, which comprises a step of performing a PCR method using the following primers (i) and (ii).
  • a forward primer and a reverse primer designed to amplify a specific region of a long DNA, a ligand-binding repurse primer having a ligand attached to the 5 ′ end of the repurse primer
  • the product amplified by the forward primer is designed to increase the 5 ′ end of the product that lacks the same number of bases in the forward primer, and a ligand is attached to the 5 ′ end.
  • a first primer set having a base sequence containing a nucleobase bound to a functional group, and (ii) a specific type of type DNA.
  • a forward primer and a reverse primer designed to amplify the region, and a ligand bound to the 5 ′ end of the forward primer.
  • the reverse primer Designed to amplify a product that lacks the same number of bases as the reverse primer at the 5 ′ end of the product amplified by the reverse primer and the reverse primer.
  • 5 'terminal ligand bound to the ligand-bound, and the above-mentioned reverse primer has a base sequence including a nucleobase bound to a functional group, and the reverse primer is A second primer set having a base sequence that is complementary to the first primer contained in the first primer set.
  • the present invention also provides: (A) a step of performing PCR using the primers (i) and (ii) below; ⁇ i) a forward ply designed to amplify a specific region of a truncated DNA; A reverse primer having a ligand bound to the 5 ′ end of the repurse primer, and a base having the forward primer on the 5 ′ end of the product amplified by the forward primer. Nucleic acids designed to increase the number of products lacking the same number of bases, including a ligand-binding phase primer having a ligand bound to the 5 'end, and wherein the forward primer is bound to a functional group.
  • a primer set of L which has a base sequence including a base; (ii) a forged primer designed to amplify a specific region of a vertical DNA; A reverse primer, a ligand-bound repurse primer with a ligand bound to the 5 ′ end of the reparse primer, and the forward primer having a 5 ′ end of the product amplified by the forward primer: ⁇ A ligand-binding phase primer that is designed to amplify a product that lacks the same number of bases and that has a ligand bound to the 5 'end.
  • FIG. 1 is a chart for explaining the DNA functional group introduction method of the present invention.
  • FIG. 2 is a chart for explaining the DNA functional group introduction method according to the second embodiment of the present invention.
  • FIG. 3 is a chart for explaining the DNA functional group introduction: ⁇ method according to the third embodiment of the present invention.
  • FIG. 4 is a chart for explaining the DNA functional group introduction method according to the fourth embodiment of the present invention.
  • Fig. 5 is a photograph showing the results of examining whether or not DNA introduced with methyl tomb is retained in the cell after DNA was transfected into the cell.
  • Fig. 6 is a photograph showing the results of a test confirming that a methyl group has been introduced at the desired site of DNA.
  • the method for introducing a DNA functional group of the present invention includes a step of performing a PCR method using the following (i) and (ii) pliesets.
  • a forward primer and a reverse primer designed to amplify a specific region of the vertical DNA, a ligand-binding repurse primer having a ligand bound to the 5 ′ end side of the repurse primer, Designed to amplify a product that lacks the same number of bases as the forward primer has on the 5 'end of the product amplified by the forward primer, and a ligand binds to the 5' end
  • a first primer set having a base sequence including a nucleobase bound to a functional group
  • a funnel deprime primer and a repurse primer designed to amplify a specific region of a long DNA, a ligand-bound forward primer having a ligand bound to the 5 ′ terminal side of the forward primer, Designed to amplify a product lacking the same base number as the salt number of the repurse primer on the 5 'end side of the product amplified by the repersptimer and
  • the reverse primer has a base sequence including a nucleobase bonded to a functional group, and the reverse dalymer is A second primer set having a base sequence complementary to the for- mation primer in the first primer set.
  • a primer having a base sequence including a nucleobase bonded to a functional group at a predetermined site is prepared.
  • the primer having a base sequence including a nucleobase bonded to a functional group at the predetermined site is a forward primer in the first primer set and a reparse primer in the second primer set.
  • the forward primer in the primer set and the reverse primer in the second primer set: 3 3 ⁇ 4 have complementary base sequences.
  • Examples of functional groups that the forward primer in the first primer set and the reverse primer in the second primer set have in the base sequence include, for example, methyl group, amide group, carboxyl group, and acetyl group.
  • a primer is usually synthesized.
  • a methyl group can be introduced into a primer by using a methylated nucleate as a substrate.
  • Primers can be designed according to the base sequence of ⁇ -type DN N.
  • the first primer set has a forward primer having a nucleate base bonded to a functional group in the base sequence.
  • this forward primer and reverse primer It is designed to amplify the region, and part of the reverse primer is a ligand-bound reverse probe with a ligand bound to its 5 'end.
  • ligands that bind to the reverse primer include biotin.
  • biotin There is no particular limitation on the method of binding the ligand to the 5 ′ end of the primer, and it can be carried out by a conventionally known method.
  • the base is used to create the biotin.
  • a modified nucleotide a base having a photon in the primer can be introduced. In the case of other ligands, the ligand was bound.
  • Ligand can be bound by creating a primer using a base.
  • a product lacking the same number of bases as the forward primer has on the 5 ′ end side of the product amplified by the forward primer is amplified.
  • a ligand-binding phase primer that is designed to have a ligand binding to the 5 'end.
  • the ligand that binds to the phase primer is the same as described above, and the production method is also different.
  • the second primer set is the river primer and the forged primer described above, and is designed to increase a specific region of the vertical DNA, and part of the forked primer is The ligand is bound to the 5 'end, and the ligand is the first binding forward primer. Examples of the U-gand that binds to the foam primer include those described above, and the manufacturing method is also as described above.
  • the second primer set is designed so as to increase the product lacking the same number of bases as the number of bases of the reverse primer at the 5 ′ end of the product amplified by the reverse primer.
  • a ligand-bound ford primer having a ligand bound to the 5 'end is included. Examples of the ligand that binds to the foam primer include those described above, and the manufacturing method is also as described above.
  • the method for introducing a DNA functional group of the present invention includes the step of performing the PCR method using (i) the first primer set and (ii) the second primer described above.
  • the DNA functional group introduction method of the present invention includes (A) the step of performing the above PCR method, and the following steps (B), (C) and (D).
  • FIG. 1 is a chart for explaining the DNA functional group introduction method of the present invention.
  • FIG. 1 illustrates the method for introducing a DNA functional group of the present invention using a vector in which a reporter gene (luciferase FP) is introduced downstream of a promoter / enhancer sequence.
  • a reporter gene luciferase FP
  • the expression of the reporter gene introduced downstream of the promoter enhancer site shows how the methyl group is introduced into the promoter / enhancer site to influence the transcriptional activity.
  • the DNA functional group introduction method of the present invention is used.
  • the DNA functional group introduction method of the present invention comprises a forward primer (1 F) and a reverse primer (2R) designed to amplify a specific region of a cage DNA.
  • a product amplified by a ligand-bound reverse primer (1 R) and a forward primer (1 F) with biotin (indicated by B in the figure) bound to the 5 'end of the repurse primer (2R) Designed to amplify a product that lacks the same number of bases as the forward primer (1 F) has on the 5 'end side of the 5' end, and that binds biotin to the 5 'end side.
  • the first primer set containing the word primer (2 F) is used.
  • the forward primer (1 F) contains a nucleobase bonded to a methyl group.
  • As the forward primer (1 F), one having about 30 to 50 bases is preferably used.
  • the DNA functional group introduction method of the present invention comprises a repurse primer (3 R) and a forward primer (4 F) designed to increase a specific region on a mirror-type DNA, and the forward primer.
  • a second primer that is designed to amplify a product that lacks the same number of salt tombs as it has and includes a ligand-bound reverse primer (4R) that is biotin linked to the 5 'end.
  • the reverse primer (3R) is used as the first primer set. It has a base sequence that is complementary to the forward primer (1 F) contained in the base and contains a nucleobase linked to a methyl group.
  • the reverse primer (3R) preferably has about 30 to 50 bases.
  • PCR is carried out using the first primer set and the second primer set (step (i)).
  • the DNA polymerase used in PCR means an enzyme that synthesizes a new DNA chain using the DNA chain as a saddle, and is not particularly limited: ⁇ , for example, pol 1 type DNA polymerase (E.
  • Bacillus caldotenax (hereinafter referred to as “B. ca”) and Bacillus stearothermophilus (hereinafter referred to as “B. st”), such as DN A polymerase derived from a bacterium belonging to the genus Bacillus and the DN A polymerase. 5′- ⁇ 3 ′ exonuclease activity mutants, etc. Furthermore, it has a strand displacement activity like the above-mentioned Kleoke fragment and has 5 ′ ⁇ 3 ′ exonuclease activity.
  • DNA polymerase is also included in the strand displacement type DNA polymerase
  • the above-mentioned DNA polymerase is a mixture of a plurality of DNA polymerases, and is not particularly limited S DNA polymerase having the above strand displacement activity and strand displacement activity It may also be a mixture of DNA polymerases that do not have any.
  • Reaction conditions in the PCR method may be general conditions. For example, after treatment at about 95 ° C. for 5 to 10 minutes, at about 95 ° C. for 15 to 30 seconds, about 55 to A cycle of 15 to 30 seconds at 60 ° C and 15 to 30 seconds at approximately 72 ° C may be 25 to 30 cycles. Above temperature and time The interval can be freely changed within the general ability of those skilled in the art in consideration of the length of the primer and the GC content.
  • Fig. 1 (b) shows the product amplified by the first primer set and the second primer set.
  • the product amplified by the forward primer (1F) in the first primer set contains a methyl group.
  • the product amplified by the ligand-bound reverse primer has a complementary base sequence to the product amplified by F 1 -primer primer (1 F), and piotin is bound to the 5 ′ end. ing.
  • the products amplified by the reverse primer (2R) and the ligand-bound forward primer (2F) 'included in the first primer set are the forward primer (1F) and the ligand-bound reverse primer (1R). ), The product is shorter by the length of the number of bases possessed by the forged primer (1 F). In addition, piotin is bound to the 5 ′ end i J of the product amplified by the ligand-bound forward primer.
  • the product amplified by the reverse primer (3R) in the second primer set contains a methyl group, and the product amplified by the ligand-bound forward primer (3F) It has a base sequence complementary to the product amplified by the Perth primer (3R), and biotin is bound to the 5 'end.
  • the forward primer (4 F) and ligand-bound repercussive primer (4R) and forward primer (4 F) included in the second primer set are fc # widened by the reverse primer (3R) and ligand-bound forward.
  • the product is shorter than the product amplified by the primer (3 F) by the number of bases unique to the reverse primer (3 R). Biotin is bound to the 5 'end of the product amplified by the ligand-bound reverse primer.
  • Step (B) is a step of removing the product amplified by the ligand-binding phase primer and the ligand-binding reverse primer contained in the first primer and the second primer set. 05006830
  • a method for removing the product amplified by the ligand-binding phase primer and the ligand-binding reverse primer a method using a ligand-binding compound can be mentioned. Specifically, the mixture of the PCR products obtained in step (A) was brought into contact with a solid phase to which a ligand-binding compound such as avidin or streptavidin was bound, and did not bind to this solid phase. A method for recovering the product may be mentioned. That is, as a combination of a ligand and a ligand-binding compound, for example, a combination of biotin-avidin or piotin-streptavidin can be mentioned.
  • the product amplified by the forward primer (1 F) has a product that is amplified by the reverse primer (2 R) and a complementary base sequence.
  • the product amplified by the reverse primer (3R) has a complementary base sequence to the product amplified by the forward primer (4 F).
  • step (C) will be described.
  • step (C) after a part of the PCR products are removed by step (B), the remaining PCR products are annealed. Annealing can be performed by cooling the solvent containing DNA, and varies depending on the GC content of the DNA obtained, for example, 85 ° C, 65 ° C, 3 ° C every 20 minutes. This can be done by gradually decreasing the temperature to 7 ° C, 20 ° C, 4 ° C.
  • complementary strands form a double strand as shown in FIG. 1 (d).
  • the forward primer (1 F) and the reperprimer (3 R) have a complementary base sequence
  • the product amplified by the forward primer (1 F) is the repurse primer (2 R).
  • the repurse primer (3 R) a product that has a complementary base sequence
  • the repurse primer (3 R) The amplified product has a complementary base sequence to the product amplified by the forged primer (4 F).
  • the ligand-bound forward primer (2 F) and the ligand-bound reverse primer (4 R) are shorter by the number of bases of the forward primer (1 F) and reverse primer (3 R), respectively.
  • step (C) when annealed in step (C), a DNA fragment having the same base sequence as that of the truncated DNA and having a methyl group introduced therein can be obtained.
  • Step (D) is a step of performing a DNA binding reaction.
  • the DNA binding reaction is a reaction that connects the unbonded DNA parts of the PCR product annealed in step (C).
  • a DNA having the same base sequence as the vertical DNA and having a methyl group introduced in a point-specific manner can be obtained (Fig. 1 (e) ).
  • DNA can be bound by DNA ligase.
  • the DNA ligase include T4 DN A ligase, E. coli DNA ligase and the like.
  • the conditions for binding DNA by DNA ligase vary depending on the DNA ligase used, and can be performed according to the optimal conditions for the DNA ligase used.
  • the DNA obtained as described above has a methyl group in the promoter no enhancer sequence, and has a reporter gene downstream of the promoter / enhancer sequence.
  • the reporter gene to be used is not particularly limited.
  • it encodes Green Fluorescent Protein (GFP), luciferase, —galactosidase, —gnorechronidase, chloramphenicol acetylenotransferase, peroxidase, etc. Include, but are not limited to, DN ⁇ ⁇ .
  • the reporter gene expression is briefly described below.
  • the point-specific DNA group-derived DNA obtained as described above is linked to an appropriate vector, and a reporter gene is expressed in an appropriate host cell, affecting the expression of this reporter gene. It is possible to examine whether there is any methylation and to investigate the effects of methylation.
  • Examples of the vector used here include plasmids derived from E.
  • coli eg!> BR322, pBR325, pUC18 or pUC118
  • Bacillus subtilis ° rasmid eg pUB110, p TP 5 or p C 1 94
  • plasmids derived from yeast eg, p SH 19 or p SH 15
  • pacteriophages such as lambda phage
  • viruses such as lettuce virus, vaccinia virus or paku mouth virus
  • ⁇ Animal viruses, A 1 to 11, p XT 1, p R c / CMV, p R c / RSV, pc DN I I ZN eo etc. are used as promoters used in the present invention.
  • T3 promoter, araBAD promoter, etc. when the host is Bacillus, SP01 promoter, penP promoter, XYL promoter, HWP promoter, CWP promoter, etc. are preferred, and when the host is Bacillus subtilis, Promoter, SP02 promoter, pen P promoter, etc. are preferred, and when the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc.
  • SR promoter When using animal cells as a host, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc.
  • polyhedrin promoter when insect cells are used as hosts, polyhedrin promoter, 0plE2 promoter, etc. are used.
  • the above-mentioned vectors include, in addition to the above, known enhancers, splicing signals, poly A addition signals, selection markers, SV40 replication origins (hereinafter sometimes abbreviated as SV40 o rgdi), etc. Can be added.
  • Examples of 2s3 ⁇ 4 host cells for expressing a vector ligated with a DNA introduced with the methyl group include, for example, Escherichia, Bacillus, yeast, and insects! 3 ⁇ 4, insect Animal cells are used.
  • Escherichia bacterium include Escherichia coli K 1 2 ⁇ DH 1 (Proc. Natl. Acad. Sci.
  • Bacillus subtilis Bacillus subtilis MI 1 14 (Gene, 24 ⁇ , 2 5 5 (1 9 8 3)), 2 0 7-2 1 [Journ al of Biochemistry, 9 5 8, 8 7 (1 9 84)] and Bacillus previs, etc.
  • yeast 3 ⁇ 4 For example, Saccaromyces cerevisiae AH 22, AH 2 2 R-, NA 8 7-1 1 A , DKD— 5 D, 2 0 B-1 2, Schizosaccaromyces pombe NCYC 1 9 1 3, NCYC 2 0 36, Pichia pastoris M 7 1 and Hansenula polymorpha, etc.
  • insect cells for example, when the virus is Ac NPV, a larvae-derived strain of night stealer Spodoptera frugiperda cells, MG 1 cells derived from the midgut of Trichoplusia ni, High Five TM cells derived from eggs of Trichop lusia ni, cells derived from Mamestra brassicae or cells derived from Estigme na acrea, etc. Force S is used.
  • the virus is B m NPV, cocoon-derived cell lines (Bombyx mori Itoda vesicles; BmN cells) and the like are used.
  • S f cells examples include S f 9 cells (ATCC CRL 1711), S f 21 cells (above, Vaughn, JL et al., In Vivo, 13, 213-217, (1977)). Etc. are used.
  • S f 9 cells ATCC CRL 1711
  • S f 21 cells above, Vaughn, JL et al., In Vivo, 13, 213-217, (1977)
  • Etc. are used.
  • the silkworm larvae force S is used [Maeda et al., Nature, 3 1 5 ⁇ , 5 9 2 (1 9 8 5)].
  • mammalian cells examples include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr-) cells) Mouse L cells, Max At T-20, mouse myeloma cells, rat GH 3 and human FL cells.
  • Mammalian cells introduced with a vector ligated with a methyl group-introduced DNA include, for example, about 5 to 20% pup serum, essential medium (MEM), Dulbecco's modified minimum essential medium. (DMEM), RPM I 160 medium and 199 medium can be cultured by adding PKA, thiazolidinedione derivative or prostaglandins.
  • the pH of the medium is preferably about 6 to 8, and the culture is usually carried out at a temperature of about 30 to 40 ° C. for about 3 to 72 hours.
  • a conventionally known method for example, using a suitable primer: the PCR method, the MPS method, and the like can be mentioned.
  • the DNA functional group introduction method of the present invention can be used for introducing a functional group into a vector in a point-specific manner. That is, the DNA functional group introduction method of the present invention can be used as a method for constructing a point-specific functional group introduction vector.
  • the forward primer (1 F) and the reverse primer (3 R) each contain one cytosine having a methyl group, but the forward primer (1 F) and the reverse primer — (3 R) may contain more than one.
  • DNA having a plurality of methyl groups can be obtained.
  • the first primer set and the second primer set are used.
  • the third primer set is further used.
  • the primer set of 3 is designed to amplify a specific region of the type DNA, and this primer set and reparatory primer A ligand-bound reverse primer in which a primer is bound to the 5 'terminal side of the reverse primer, and the forward-primer side of the product amplified by the forward primer.
  • the forward primer is a nucleus bound to a functional group
  • the forward primer is a primer set having a base sequence complementary to the repurse primer included in the first primer set.
  • the 5 'end of the reverse reverse primer contained in the first primer set is connected to the third primer set. It is preferable to measure H so as to have a product that lacks the same number of bases as the number of base primers contained in the primer set.
  • FIG. 2 is a chart for explaining the D IN A functional group introduction method according to the second embodiment of the present invention.
  • a third set of primers (5F, 5R, 6F and 6R) is used.
  • a forod primer designed to amplify a specific region of a truncated DNA.
  • 5 F and the reverse primer (5), the ligand-bound reverse probe (6 R) with the ligand bound to the 5 ′ end of the reverse primer (5 R), and the forward primer (5 F) It is designed to amplify a product that lacks the same number of bases Ifc as the forward primer (5 F) at the 5 'end of the product amplified and A ligand-bound forward primer (6 F) bound to a carrier, and the forward primer (5 F) has a base sequence containing a nucleobase bound to a functional group and ferries, and the forward Primer (5F) is the first plastic It has a nucleotide sequence complementary to the Reverse primer (2R) contained in Mase' bets, containing f this nucleobase bound to a methyl group.
  • the reverse primer 2R included in the first primer set is It is designed to have a product that lacks the same number of bases as the forward primer (6 F) contained in the primer set of 3.
  • the PCR method in step (a) is performed using the first primer set, the second primer set, and the third primer set. (See Figure 2 (a)).
  • the PCR product shown in Fig. 2 (b) can be obtained by performing the PCR reaction in step (a). Next, perform the same operations as described above, and remove the ligand-bound forward primer and the ligand-bound reverse primer (see Fig. 2 (c)), and then anneal (see Fig. 2 (d)). Perform a binding reaction of, and (see Fig. 2 (e)) to obtain DNA with a functional group (methyl group) introduced.
  • a functional group methyl group introduced.
  • the methyl group is also introduced into the reporter gene region.
  • a functional group (methyl group) is introduced into the promoter / enhancer region and the reporter gene region, and in the same manner as shown in FIG. It can be used as a means to investigate the effect on transcription caused by the introduction of a functional group.
  • the functional grave is a methyl group, but in the present invention, an amide group or the like can be used as the functional group. Therefore, the introduced amide group (for example, the amide group bound to the promoter noenhashi region in FIG. 2) is bound to, for example, a compound that is an anticancer agent and separated from the amide group. Signal peptides that bind to a specific group of cells or subcellular organelles (eg, the amide group bound to the region of the reporter ft gene in Figure 2) bound to a position.
  • a specific group of cells or subcellular organelles eg, the amide group bound to the region of the reporter ft gene in Figure 2 bound to a position.
  • a signal protein specific for cancer cells, a signal peptide specific for mitochondria, and a signal protein for introducing the protein into the nucleus are combined to form a nucleotide-protein-drug complex.
  • the drug moves into cancer cells, mitochondria, and the nucleus, which is effective in treating cancer.
  • a drug used for forming the complex a drug conventionally used for the treatment of cancer can be used without particular limitation.
  • drugs used for the treatment of specific diseases can be used.
  • non-atypically proteins having affinity for mammalian cells are commercially available (for example, “Voige 1 protein” manufactured by Invitrogen, Inc.).
  • a functional group is introduced into a specific site of DNA, and an arbitrary distance from the specific site is introduced.
  • Other functional groups can be introduced at the site.
  • a fourth primer set, a fifth primer set, and the like can be further used.
  • the primer included in the fourth primer set has the same relationship with the third primer set as the first primer set and the third primer set.
  • the fourth primer set it is possible to introduce functional groups into the three force sites that are located away from each other.
  • the method for introducing a DNA functional group according to the third embodiment of the present invention includes a step of performing a PCR method, amplification using a ligand-binding phase primer and a ligand-combining repurse primer.
  • the process for removing the produced product is the same as the method shown in Fig. 1 & Fig. 3 (see Fig. 3 (a) and (b)). In the method shown in FIG.
  • the forward primer (the first primer set included in the first primer set ( Product amplified by 1 F) and included in the second primer set
  • a unique gene sequence such as a restriction enzyme recognition site, is repeated twice over the 3 'end of the product amplified by the reverse primer (3R).
  • the Not I recognition site is amplified at the 3 'end of the product amplified by the first forward primer (1 F) and amplified by the reverse primer (3 R) included in the second primer set.
  • An Asc I recognition site is bound to the 3 'end of the resulting product.
  • a restriction enzyme recognition site is bound to the 3 'end of the resulting product, but the reverse primer (2R) included in the first primer set and the 5' of the first primer set included in the second primer set. It may be bonded to the terminal side.
  • a Not I recognition site and an Asc I recognition site are used as restriction enzyme recognition sites.
  • the present invention is not limited to these, and any restriction enzyme recognition site is used. May be.
  • FIG. 3 (c) since the restriction enzyme recognition site is bound to the terminal, it is shown in FIG. 3 (e). Thus, it will have a loop structure at the end.
  • FIG. 4 is a chart for explaining the DNA functional group introduction method according to the fourth embodiment of the present invention. As shown in FIG.
  • the method for introducing a DNA functional group according to the third embodiment of the present invention includes a step of performing a PCR method, and a product amplified by a ligand-binding forward primer and a ligand-binding reverse primer.
  • the removal process is the same as the method shown in FIG. 1 (see FIGS. 4 (a) and (b)).
  • FIG. 4 after removing the product amplified by the ligand-bound forward primer and the ligand-bound reverse primer, The product amplified by the forward primer (1 F) included in the first primer set and the reverse primer (3 R) included in the second primer set.
  • the restriction enzyme recognition site is bound to the 3 'end of the product in such a combination that it can form a base pair when approaching each other.
  • the product amplified by the first forward primer (1 F) included in the first primer set and the reverse primer (3 R) included in the second primer set are used.
  • a restriction enzyme recognition site is attached to the 3 'end of the amplified product, but the reverse primer (2R) included in the first primer set and the forward primer included in the second primer set 5 'It may be attached to the terminal side.
  • the operation is performed in the same manner as shown in FIG. In other words, annealing is performed (see Fig. 4 (d)), and DN A binding reaction is performed (see Fig. 4 (e)) to obtain DN A into which a functional group (methyl group) has been introduced.
  • a functional group such as a methyl group
  • the DNA functional group introduction method of the present invention introduces a functional group such as a methyl group into the transcriptional regulatory region of DNA, and examines the influence on the transcription caused by the introduction of the functional group. It can be used as a means.
  • a promoter region having a methyl group is linked in a position-specific manner upstream of a reporter gene such as luciferase, ⁇ -galactosidase, ⁇ -darc-mouthed nidase, chloramphenicol acetyltransferase, peroxidase, Transcription can be performed to examine the effect on transcription.
  • a reporter gene such as luciferase, ⁇ -galactosidase, ⁇ -darc-mouthed nidase, chloramphenicol acetyltransferase, peroxidase, Transcription can be performed to examine the effect on transcription.
  • DNA introduced with a functional group such as an amide group by the DNA functional group introduction method of the present invention has an ability to bind to a protein, and forms a protein-DNA complex. This complex can be used to detect the interaction between a DNA-binding protein and DNA.
  • a DNA into which a functional group has been introduced by the DN ⁇ functional group introduction method of the present invention is transcribed.
  • D ⁇ A transgene
  • This transgene is used as a vector in which a resistance gene exhibiting resistance to a specific compound such as neo is combined with a gene having a methyl group in a position-specific manner.
  • This transgene is amplified by inserting an appropriate expression vector, and after amplification, the gene fragment is excised, and the excised transgene is introduced into, for example, a pronuclear fertilized egg of a rat by a microinjection method. After the introduction, the fertilized egg is transplanted to a temporary parent and laid. Whether or not the PBSVT transgene has been introduced into the pups born can be confirmed by extracting DNA from a portion of the sample (eg, the tip of the tail) and then using Southern plot analysis or PCR analysis. The individual confirmed to have the transgene is the 3 ⁇ 4 founder (Founder), and this primary rat is mated with a healthy wild-type rat.
  • Founder 3 ⁇ 4 founder
  • the F 1 rat confirmed to have this transgene introduced is crossed with a healthy rat.
  • a model rat strain in which the expression of a specific gene is controlled can be established.
  • cancer can be treated by introducing such a DNA gf fragment into the cell. . That is, the DN having a functional group introduced into the transcriptional regulatory region of a cancer gene can be used as a prophylactic or therapeutic agent for cancer by the method of introducing a NA functional group of the present invention. Examples of methods for the prevention and treatment of this cancer prevention method include a method using a non-viral vector and a method using a viral vector. Such an administration method will be briefly described below.
  • the above-mentioned DNA can be introduced into cells or tissues by the following method using a recombinant vector in which the above-mentioned DNA is incorporated into a conventional gene expression vector.
  • Examples of the method for introducing a gene into a cell include a phosphate-calcium coprecipitation method; a DNA direct injection method using a micro glass tube.
  • Examples of gene transfer methods to tissues include gene transfer methods using internal type liposomes, gene transfer methods using electrostatic type liposomes, HV J -ribosome method, and improved HV J—.
  • Ribosome method HVJ-AVE liposome method
  • receptor-mediated gene transfer method method of transferring D3 A molecule into cells together with carrier (metal particles) with particle gun, direct method of naked-DNA transfer, positive charge
  • Examples thereof include a polymer introduction method.
  • expression vectors used in the above-described method include p CAGGS (Gene 108, 193-200 (1991)), p BK-CMV, pc DN A 3.1, p Z eo SV (Invitrogen Corporation, S (Tratagene)).
  • examples of the viral vector include feed-changing adenovirus and retrovirus. More specifically, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, ⁇ kushina uinores, boxwii / res, polioui> ⁇ res, shinbisui / res, sendaiquinores, SV40, immunodeficiency virus
  • a DNA virus such as (HIV) or RNA Winoles
  • adenovirus infection efficiency is known to be much higher than when other virus vectors are used. Therefore, it is preferable to use an adenovirus vector system.
  • the DNA obtained by the method of the present invention to which a point-specific functional group has been added can be used as a vector for transporting the sequence-specifically by combining a drug with the functional group.
  • the bond between DNA and peptide is a bond that DNA and peptide are not easily released, for example, a covalent bond.
  • This DNA-peptide bond f is used for, for example, a region-specific DNA cleaving enzyme.
  • a cell-specific gene therapeutic vector can be obtained by binding a protein specifically taken up into a certain cell to a functional group site of a therapeutic DNA vector.
  • a DNA repair capacity quantification system can be applied to a method for screening for the occurrence of cancer caused by smoking. In smoking, it is known that an alkyl group is added to a gene, and the gene site to which an alk / re group is added subsequently mutates to cause cancer. Normally, this mutation is repaired, but if this ability is low, there is a high probability of smoking causing cancer.
  • the above DNA repair capacity quantification system can be used as a system for measuring the repair rate of mutations caused by the addition of alkyl groups by smoking.
  • the method of the present invention can be used for the creation of mutants using efficient point mutation oligonucleotides in an in vivo system that utilizes mismatch repair to thymine bases. For example, when an alkyl group is added to guayun, thymine is often substituted during repair. Using this property, it can be used as a method for constructing a point-specific mutation system in vivo by adding a guanine base alkyl group at any position.
  • peptide tag-specific antibodies can be used for detection of specific nucleotide sequences.
  • the method of the present invention can be used to produce a three-dimensional structure by linking alkyl groups & thus forming a cross-link of single-stranded DNA. That is, end alkyl groups have the property of being bonded to each other. Using this property, any position of the oligonucleotide It can be used for facilitating cell-to-cell transfer by adding an alkyl group to the nucleotide at the position to form a solid structure by forming a single-stranded DNA bridge.
  • the CMV promoter 1 to GFP part of the pEGFP-N1 vector manufactured by Clontech Co., Ltd. was cut out and incorporated into the PUC18 vector to prepare PUC / EGFP plasmid 3 ⁇ 4r.
  • a PCR method was performed using this plasmid and the following primers to increase the Green Fliiorescent Protein (GFP) gene.
  • GFP Green Fliiorescent Protein
  • Reverse primer 5 '-biot-agcggataacaatttcacacagga- S' (Major (J number: 4) Second primer set
  • the reaction conditions for PCR are as follows.
  • a forma primer (5 -P-gggtggtgcccatcctggtc,) having the base sequence represented by SEQ ID NO: 9 gagctggac (m) ggc ⁇ gacgtaaacggccaca-3), and in place of the reverse primer having the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 1 0
  • the DNAs obtained in Examples 1 and 2 and Comparative Example 1 were transfected into cells, and it was examined whether or not DNA introduced with a methyl group was retained in the cells.
  • the recombinant vectors obtained in Examples 1 and 2 and Comparative Example 1 were transformed into C0S7 cells by the lipofection method to prepare transformants. After the obtained transformant was cultured, genomic DNA was extracted using a Qiagen DNA purification kit. The obtained DNA was subjected to PCR using a primer having the base sequence represented by SEQ ID NO: 17 and a primer having the base sequence represented by SEQ ID NO: 18.
  • PCR reaction conditions are as follows.
  • Fig. 5 (a) shows the results obtained using a primer having the base sequence represented by SEQ ID NO: 17 and a primer having the base sequence represented by SEQ ID NO: 18.
  • Fig. 5 (b) The result obtained using a primer having the base sequence represented by SEQ ID NO: 18 and the T lymer and the base sequence represented by SEQ ID NO: 6 is shown.
  • Lane 1 is the DNA obtained in Comparative Example 1
  • Lane 2 is the DNA obtained in Example 1
  • Lane 3 is the DNA obtained in Example 2
  • Lane 4 and Lane 5 are the results.
  • Lane 6 is 28
  • Genomic DNA was extracted from the vector-introduced cells using Qiagen's DNAffr kit.
  • the sample thus prepared was converted from cytosine to uracil by bisulfite. That is, the sample was sulfonated, hydrolyzed, and then alkali desulfonated. Alkaline desulfonation! On the finished sample
  • NaOH was added to a final concentration of 0.3 N and incubated at 37 ° C for 15 minutes.
  • bisulfate treatment methylated cytosine remains unconverted.
  • a PCR method was performed using the following primers, and it was confirmed that a desired DNA (a methyl group was introduced at the O site).
  • a primer having the base sequence represented by SEQ ID NO: L 3 and a primer having the rot group sequence represented by SEQ ID NO: 15 are used. It was. In addition, the fact that it is not methylated can be confirmed by using a primer having a base sequence represented by SEQ ID NO: 14 and a primer having a base sequence represented by SEQ ID NO: 15. In addition, as a control, a PCR reaction was performed using a primer having the base sequence represented by SEQ ID NO: 15 and a primer having the base sequence represented by U number: 16.
  • Fig. 6 shows the results of electrophoresis.
  • Fig. 6 is a photograph showing the results of a test to confirm that a methyl group has been introduced at the desired site of DNA.
  • lane 1 is the result when a PCR reaction was performed using a primer having the base sequence represented by SEQ ID NO: 15 and a primer having the base sequence represented by SEQ ID NO: 16;
  • Lane 2 shows the results when PCR reaction was performed using a primer represented by SEQ ID NO: 13: a primer having a base sequence and a primer having a base sequence represented by SEQ ID NO: 15;
  • 3 shows the results when PCR was performed using a primer having the base sequence represented by SEQ ID NO: 14 and a primer having the base sequence represented by SEQ ID NO: 15; It is a molecular weight marker.

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Abstract

L’invention concerne un procédé permettant d’introduire un groupe fonctionnel à ADN à l’aide d’un procédé PCR en utilisant les amorces (i) et (ii) suivantes : (i) un premier ensemble comprenant une amorce sens destinée à amplifier une région spécifique (présentant une nucléobase se liant à un groupe fonctionnel) et une amorce anti-sens, une amorce anti-sens de liaisonDe liaison à un ligand ayant un ligand en position terminale en 5' de cette dernière ainsi qu’une amorce sens de liaison à un ligand qui amplifie un produit, dans lequel un nombre de bases identique à celui de la première amorce a été supprimé au niveau du groupe terminal en 5'du produit amplifié par la première amorce, et a un ligand en position terminale en 5' ; (ii) un deuxième ensemble comprenant une amorce sens destinée à amplifier une région spécifique et une amorce anti-sens (ayant une nucléobase se liant à un groupe fonctionnel et une séquence complémentaire à l’amorce sens du premier ensemble), une amorce sens de liaison à un ligand ayant un ligand en position terminale en 5' et une amorce anti-sens de liaison à un ligand qui amplifie un produit dans lequel un nombre de bases identique à celui présent dans cette dernière a été supprimé au niveau du groupe terminal en 5' du produit amplifié par cette dernière et a un ligand en position terminale en 5'.
PCT/JP2005/006830 2005-03-31 2005-03-31 Procede d’elaboration d’un vecteur permettant d’introduire un groupe fonctionnel specifique a un point Ceased WO2006114834A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn
JP2002524473A (ja) * 1998-09-09 2002-08-06 ジエンザイム コーポレイション プラスミドベクターのメチル化
JP2002543801A (ja) * 1999-05-10 2002-12-24 スミスクライン・ビーチャム・コーポレイション 修飾された核酸分子の産生法
JP2003515344A (ja) * 1999-12-06 2003-05-07 インスティチュート・パスツール エピジェネティックな制御モチーフ含有量が減少または増加した単離ヌクレオチドおよびその使用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn
JP2002524473A (ja) * 1998-09-09 2002-08-06 ジエンザイム コーポレイション プラスミドベクターのメチル化
JP2002543801A (ja) * 1999-05-10 2002-12-24 スミスクライン・ビーチャム・コーポレイション 修飾された核酸分子の産生法
JP2003515344A (ja) * 1999-12-06 2003-05-07 インスティチュート・パスツール エピジェネティックな制御モチーフ含有量が減少または増加した単離ヌクレオチドおよびその使用

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