CN102382851A

CN102382851A - Cloning vector

Info

Publication number: CN102382851A
Application number: CN2011102645375A
Authority: CN
Inventors: 棚濑智雄
Original assignee: Takara Bio Inc
Current assignee: Takara Bio Inc
Priority date: 2010-09-03
Filing date: 2011-09-01
Publication date: 2012-03-21
Anticipated expiration: 2031-09-01
Also published as: JP2012143226A; CN102382851B

Abstract

The present invention provides a TA cloning vector, a cloning method using the vector, a manufacturing method for the vector and a kit containing the vector, wherein the TA cloning vector controls the insertion direction of the DNA fragment of PCR augmentation products, and simultaneously can easily select and insets the purposed DNA garments for cloning. The present invention provides a vector, wherein the tail end of the vector is provided with an activity-lost drug-fast gene with the nucleic acid sequence deficient, and the nucleic acid sequence codes the amino acid of the C tail end portion obbligato in the express process of the drug-fast performance of the drug-fast gene products and the amino acid of the N tail end portion. The present invention also provides a cloning method using the vector, the manufacturing method for the vector and a kit containing the vector.

Description

Cloning vector

Technical field

The present invention relates to be suitable for the method for manufacture of the carrier of PCR product cloning, the cloning process that uses this carrier, this carrier and the test kit that contains this carrier.

Background technology

Up to now, as the separation method of goal gene is known the method for utilizing PCR arranged.In the method, at first contain DNA or its fragment of goal gene, the PCR product that obtains is assembled into the clone with in the plasmid vector, thereby further use this plasmid vector to make the host transform the single bacterium colony that forms transformant through pcr amplification.Next, by the refining plasmid of formed single bacterium colony, the dna fragmentation that is inserted in the plasmid is confirmed.Through doing like this, can obtain being inserted with goal gene or its segmental plasmid.

In the used enzyme of PCR method, exist such side reaction active in the known representational Taq archaeal dna polymerase: 3 of the DNA product after duplicating ' end adds 1 Desoxyadenosine (dA) residue with the dependent mode of non-template.Therefore, use the major part of the double chain DNA fragment have the active archaeal dna polymerase amplification of such side reaction to have the 3 ' distal process that has added 1 Desoxyadenosine residue and go out structure.

Developed the TA PCR cloning PCR that utilizes the Taq archaeal dna polymerase and utilize open loop carrier (T carrier), wherein said open loop carrier has outstanding 1 deoxythymidine (dT) residue (non-patent literature 1) at two 3 ' ends.Through adopting the TA PCR cloning PCR, can under the situation that the Restriction Enzyme that does not carry out the PCR product is handled and terminal smoothing is handled, obtain being inserted with the recombinant plasmid of PCR product.

Yet there is shortcoming in traditional T A PCR cloning PCR.In the TA clone, in containing the transformant discriminating that is inserted with the segmental plasmid of PCR, utilize the α-Hu Bu property of beta-galactosidase gene usually, that is, so-called indigo plant is screened in vain.Yet, have following situation sometimes: when inserting the PCR fragment through reading frame, cause expression as the α peptide of fusion rotein with respect to base sequence derived from the α peptide gene of beta-galactosidase enzymes.In this case, cause appearing the generation of weak blue false negative bacterium colony.

In addition, in common TA PCR cloning PCR, can not control the directivity that is inserted into the goal gene in the carrier.Made modified form T-carrier in order to eliminate this shortcoming.For example; The primer pair and the combined method of T-carrier that will generate first restriction enzyme site in patent documentation 1, have been disclosed, wherein the said T-carrier of design like this: only using said primer to occur second restriction enzyme site to along reverse insertion pcr amplification product the time.In the method, use the Restriction Enzyme that cuts off second restriction enzyme site only to cut off the plasmid along reverse insertion, can only obtain such transformant thus: it maintains the plasmid that is inserted with dna fragmentation along desired orientation.Yet in the method, need further carry out Restriction Enzyme after the ligation and handle, it is miscellaneous that operation becomes.

Recently, the someone has reported such method: to generate the false negative bacterium colony in the TA PCR cloning PCR in order being controlled at, to utilize lethal gene as selective marker (non-patent literature 2).Yet, in the method, can not control the directivity that is inserted into the goal gene in the carrier.

Know that by above still do not have such cloning vector at present, it controls the direction of insertion of the such dna fragmentation of pcr amplification product easily, can easily select only to be inserted with simultaneously the clone of target DNA fragment.

The prior art document

Patent documentation

Patent documentation 1: No. 2005/021747 pph of International Publication

Non-patent literature

Non-patent literature 1:BioTechniques, nineteen ninety-five, the 19th volume, the 1st phase, 34-35 page or leaf

Non-patent literature 2:Molecular Biology Reports, 2009, the 36th volume, the 7th phase, 1793-1798 page or leaf

Summary of the invention

The problem that invention will solve

The purpose of this invention is to provide the method for manufacture of the TA cloning vector that has overcome above-mentioned shortcoming, the cloning process that uses this carrier, this carrier and the test kit that contains this carrier.

Solve the means of problem

The inventor finds; Thereby can solve above-mentioned problem the present invention by means of such carrier accomplishes; This carrier has disposed the loss of activity drug resistance gene of nucleotide sequence disappearance endways, and wherein said nucleotide sequence is encoded to the amino acid of indispensable C-terminal part in the resistance performance of drug resistance gene product or the amino acid of N-terminal part.

That is, the present invention relates to:

[1] a kind of carrier; It is the cloning vector that is formed by straight chain shape double-stranded DNA; Said straight chain shape double-stranded DNA has outstanding 1 deoxythymidine or uridine residue at two 3 ' ends; This carrier is characterised in that; It maintains the nucleic acid that the partial sequence of drug resistance gene product is encoded, and said nucleic acid is the loss of activity drug resistance gene that has lacked such nucleotide sequence, and this nucleotide sequence is encoded to the amino acid of indispensable C-terminal part in the resistance performance of said drug resistance gene product or the amino acid of N-terminal part; In addition; Said nucleic acid is configured in the said double-stranded DNA end of any one, makes when the insertion dna fragmentation that contains the nucleotide sequence that is lacked in the said loss of activity drug resistance gene at terminal portions wherein is connected to carrier, has constituted the nucleic acid that the drug resistance gene product that not have to lack is encoded.

[2] carrier of record in above-mentioned [1], wherein, said loss of activity drug resistance gene is the loss of activity drug resistance gene that has lacked such nucleotide sequence, this nucleotide sequence is encoded to the amino acid of the C-terminal part of drug resistance gene product.

[3] carrier of record in above-mentioned [2], wherein, said loss of activity drug resistance gene is made up of the nucleotide sequence that the aminoacid sequence that has lacked the amino acid whose β-Nei Xiananmei of C-terminal is encoded.

[4] carrier of record in above-mentioned [3], wherein, said loss of activity drug resistance gene is made up of the nucleotide sequence that the aminoacid sequence of record in the sequence number 17 of sequence table is encoded.

[5] carrier of record in above-mentioned [1] also comprises the 2nd drug resistance gene, and said the 2nd drug resistance gene illustrates the resistance to medicament different with aforementioned drug resistance gene.

[6] carrier of record in above-mentioned [1], it is by forming by the formed DNA of nucleotide sequence of record in the sequence number 5 of sequence table with by the formed DNA of nucleotide sequence that puts down in writing in the sequence number 18 of sequence table.

[7] a kind of method of cloned DNA may further comprise the steps (a)～(d): be template (a) with DNA, and the step of using first primer, second primer relative and archaeal dna polymerase to carry out PCR with first primer; (b) will be connected with the carrier of each record in above-mentioned [1] to [6] through the PCR product that step (a) obtains, obtain the step of cyclic DNA; (c) step that adopts the cyclic DNA obtain by step (b) host cell to be changeed shape; And (d) utilize such medicament to carry out medicament and select; Selection has the step of the transformant of cyclic DNA thus; Wherein said medicament is not have the drug resistance gene product of disappearance to chemical sproof medicament it illustrate, and said cyclic DNA forms through the PCR product is inserted in the carrier along desired orientation; Wherein, First primer has and aforementioned DNA complementary nucleotide sequence at 3 ' end side; And when the loss of activity drug resistance gene in the carrier of record in above-mentioned [1] to [6] is when having lacked the gene of the nucleotide sequence that the C-terminal amino acid partly of drug resistance gene product is encoded; Said first primer has the nucleotide sequence that is lacked at 5 ' end side; Loss of activity drug resistance gene in above-mentioned [1] to [6] in the carrier of record is when having lacked the gene of the nucleotide sequence that the amino acid of the N-terminal part of drug resistance gene product is encoded, and said first primer has at 5 ' end side and removed 5 from the complementary strand sequence of the nucleotide sequence that lacked ' nucleotide sequence behind the terminal deoxythymidine.

[8] cloning process of above-mentioned [7], wherein the archaeal dna polymerase in the step (a) has the activity of adding 1 Desoxyadenosine base at 3 of reaction product ' end with the dependent mode of non-template.

[9] cloning process of above-mentioned [7] wherein, also is included in the step that 3 of reaction product ' end adds 1 Desoxyadenosine base in step (a).

[10] a kind of method of manufacture of carrier; Comprise the following steps: to cut off the step of the circular double stranded DNA of the nucleotide sequence of record in the sequence number 19 with sequence table through Restriction Enzyme PmaCI, thereby and through having two 3 of the double-stranded DNA of the active enzyme of terminal deoxynucleotidyl transferase after cut-out ' terminal step of adding the carrier that outstanding 1 deoxythymidine or uridine residue obtain putting down in writing [1] in.

[11] a kind of test kit, it uses test kit for the clone, contains any described carrier and dna ligase in [1] to [6].

The invention effect

According to the present invention, a kind of like this carrier can be provided, promptly can only select the clone who is inserted with goal gene along desired orientation through simple operations.In addition, according to the present invention, when the transformant that contains the plasmid that is inserted with goal gene is identified, do not need X-Gal or IPTG etc., thereby simplified beamhouse operation.In addition, carrier of the present invention also plays a role in the clone that the dna fragmentation that is rich in AT is carried out.

Description of drawings

Fig. 1 is for illustrating embodiment 2 (1) the middle figure that clone the bacterium colony PCR result of the transformant that obtains through the TA that has used carrier of the present invention.

Fig. 2 is for illustrating embodiment 2 (1) the middle figure that clone the bacterium colony PCR result of the transformant that obtains through the TA that has used traditional T-carrier.

Fig. 3 is for illustrating embodiment 2 (3) the middle figure that clone the bacterium colony PCR result of the transformant that obtains through the TA that has used carrier of the present invention.

Fig. 4 is for illustrating embodiment 2 (3) the middle figure that clone the bacterium colony PCR result of the transformant that obtains through the TA that has used traditional T-carrier.

Fig. 5 is the figure that the structure of pUC19-bla Δ W 290-T DNA is shown.

Embodiment

(1) cloning vector of the present invention

Cloning vector of the present invention is made up of straight chain shape double-stranded DNA; Said straight chain shape double-stranded DNA has outstanding 1 deoxythymidine or uridine residue at two 3 ' ends; This carrier maintains the nucleic acid that the partial sequence of drug resistance gene product is encoded; Said nucleic acid is the loss of activity drug resistance gene that has lacked such nucleotide sequence; This nucleotide sequence is encoded to the amino acid of indispensable C-terminal part in the performance of the resistance of said drug resistance gene product or the amino acid of N-terminal part, and in addition, said nucleic acid is configured in the said double-stranded DNA end of any one; Make will be wherein the terminal portions insertion dna fragmentation that contains the nucleotide sequence that is lacked in the said loss of activity drug resistance gene when being connected to carrier, constitute the nucleic acid that the drug resistance gene product that not have to lack is encoded.Among this paper, so-called " disappearance " is meant in being configured in the terminal drug resistance gene of said straight chain shape double-stranded DNA, only maintains the nucleotide sequence of being encoded in other zone outside the indispensable part in the resistance performance.

So-called " said nucleic acid is configured in the said double-stranded DNA end of any one; make when the insertion dna fragmentation that contains the nucleotide sequence that is lacked in the said loss of activity drug resistance gene at terminal portions wherein is connected to carrier; constitute the nucleic acid that the drug resistance gene product that not have disappearance is encoded " is meant: the end that lacks side for the nucleotide sequence that makes said loss of activity drug resistance gene becomes in the said double-stranded DNA end of any one, and said nucleic acid is as above disposed.Yet, dispose in the double-stranded DNA end of said loss of activity drug resistance gene side 3 ' terminal outstanding 1 deoxythymidine or uridine residue, to constitute the part of said loss of activity drug resistance gene or its complementary strand.Dna fragmentation through will partly containing the partial sequence of the nucleotide sequence that lacks in the said loss of activity drug resistance gene endways is connected to cloning vector of the present invention, has constituted the nucleic acid that the drug resistance gene product that does not have disappearance is encoded.

Unqualified to giving to the chemical sproof said drug resistance gene of particular agent, as long as it is encoded to making the host that the medicament that suppresses host's growth is had chemical sproof protein.For example, can use the chemical sproof gene of paying to Ampicillin Trihydrate, paraxin, Oxacyclotetradecane,erythromycin deriv, qingfengmeisu qiong, kantlex, Xin Meisu, Totomycin, spectinomycin, Streptomycin sulphate, tsiklomitsin etc. among the present invention.Preferably, can use anti-Ampicillin Trihydrate property gene (β-Nei Xiananmei gene) among the present invention, more preferably can use from colibacillary β-Nei Xiananmei gene.

As in said loss of activity drug resistance gene, lack and in the performance of the resistance of drug resistance gene product the amino acid of indispensable C-terminal part or the amino acid of N-terminal part; Can enumerate: preferred 1～5 amino-acid residue of indispensable C-terminal or N-terminal from the performance of the resistance of drug resistance gene product; More preferably 1～3 amino-acid residue of indispensable C-terminal or N-terminal from the performance of the resistance of drug resistance gene product, the amino-acid residue of indispensable C-terminal or N-terminal in the resistance performance of further preferred drug resistance gene product.In addition; In this specification sheets; So-called " to the nucleotide sequence that in said loss of activity drug resistance gene, lacks and the amino acid of the amino acid of indispensable C-terminal part in the resistance performance of drug resistance gene product or N-terminal part is encoded "; Be not limited to comprise those nucleotide sequences of all codons that such amino acid is encoded; So long as losing in the chemical sproof scope of drug resistance gene product, can be to have lacked those nucleotide sequences to a part of nucleotide sequence of its codon of encoding for an amino acid in the above-mentioned amino acid owing to this disappearance.For example; Have following situation: in said loss of activity drug resistance gene, lack and in the performance of the resistance of drug resistance gene product the amino acid of indispensable C-terminal part or the amino acid of N-terminal part be the phenylalanine(Phe) of C-terminal; To its codon of encoding be 5 '-TTC-3 ', simultaneously wherein 5 '-also nucleotide sequence of TC-3 ' in said loss of activity drug resistance gene, lacking and the amino acid of the amino acid of indispensable C-terminal part in the resistance performance of drug resistance gene product or N-terminal part is encoded.As the nucleotide sequence that in said loss of activity drug resistance gene, lacks and the amino acid of the amino acid of indispensable C-terminal part in the performance of the resistance of drug resistance gene product or N-terminal part is encoded; Among the present invention it is not limited especially, but the nucleotide sequence of preferably the C-terminal amino acid partly of drug resistance gene product being encoded.As the loss of activity drug resistance gene of such nucleotide sequence disappearance, can enumerate the nucleotide sequence that the such aminoacid sequence to the β-Nei Xiananmei of C-terminal aminoacid deletion of nucleotide sequence shown in the sequence number 17 of (for example) sequence table is encoded.

The carrier that contains nucleotide sequence shown in the sequence number 17 of ordered list for example can be by following method preparation.With 5 '-CATTGG-3 ' modification become as the PmaCI Restriction Enzyme cut off 5 of position '-CACGTG-3 '; Wherein 5 '-CATTGG-3 ' is to the 290th amino acids residue (tryptophan residue as the β-Nei Xiananmei C-terminal; Hereinafter is labeled as W290 sometimes) and the 289th amino acids residue (histidine residues, hereinafter are labeled as H289 sometimes) of its front nucleotide sequence of encoding.Polypeptide by the modification β-Nei Xiananmei coded by said gene for preparing like this can not decompose β-Nei Xiananleikangshengsu (for example Ampicillin Trihydrate).When wherein being inserted with this modification β-Nei Xiananmei gene and in the zone beyond the said gene, not having the carrier of PmaCI place of incision with the PmaCI cut-out, the β-Nei Xiananmei gene that has generated the sequence deletion of coding W290 is positioned at terminal straight chain shape double-stranded DNA.In addition; Utilize two 3 ' ends of the double-stranded DNA that currently known methods adds 1 deoxythymidine (perhaps uridine) residue as above to obtain to, can prepare the suitable straight chain shape carrier that is connected with the PCR product that has 1 deoxyadenosine residue at 3 ' end thus.

At this; Though the nucleotide sequence shown in the sequence number 17 of use sequence table is illustrated; But straight chain shape carrier of the present invention is not limited to above-mentioned object lesson; Through C-terminal part or the N-terminal aminoacid sequence partly of considering drug resistance gene, and suitably select to use known Restriction Enzyme, same method capable of using prepares straight chain shape carrier.

As carrier of the present invention, to its not special qualification, can use plasmid vector, phage vector, phagemid carrier among the present invention.As plasmid vector, can use the plasmid vector that maintains the replication orgin (in the intestinal bacteria situation, being pUC ori, pACYCori etc.) that in used host, plays a role.In carrier of the present invention, except expressing the promoter sequence that the drug resistance gene carried uses, also can contain promoter sequence that the genetic expression on the dna fragmentation that (for example) control cloned uses and operon sequence etc.In addition, carrier of the present invention can also have the 2nd drug resistance gene, and it illustrates the resistance to medicament different with aforementioned drug resistance gene.Using under the situation of (for example) anti-Ampicillin Trihydrate property gene as aforementioned drug resistance gene; As the 2nd drug resistance gene; Among the present invention it is not limited especially; Can enumerate the chemical sproof gene of paying to paraxin, Oxacyclotetradecane,erythromycin deriv, qingfengmeisu qiong, kantlex, Xin Meisu, Totomycin, spectinomycin, Streptomycin sulphate, tsiklomitsin etc., wherein preferably enumerate anti-kantlex property gene.As the preferred example of carrier of the present invention, can enumerate carrier with structure shown in Figure 5.As the further object lesson of carrier of the present invention, can enumerate the carrier that DNA that the nucleotide sequence put down in writing in the sequence number 18 by the DNA that forms of nucleotide sequence of record in the sequence number 5 of sequence table and sequence table forms forms.

Through carrier of the present invention being used for cloning process of the present invention described below, occurring the false positive clone in the time of can suppressing to obtain transformant, and can improve the acquisition efficient of positive colony.In this specification sheets; So-called " the acquisition efficient of positive colony " be meant keep being inserted with expectation insert segmental cyclic DNA expect in clone's scope; In fact kept inserting the shared ratio of clone of the segmental cyclic DNA of expectation insertion; Wherein said expectation insert fragment be through with the corresponding screening method of carrier that in the TA clone, uses (blue white screening or medicament selection etc.), select the transformant that obtains from transforming through the cyclic DNA that obtains by the TA clone.The measuring method of the acquisition efficient of positive colony can be measured according to the method for record among the application embodiment 2 to 4.

In addition, through carrier of the present invention being used for cloning process of the present invention described below, can obtain inserting the cyclic DNA that fragment is only inserted along any direction.

(2) cloning process of the present invention

Cloning process of the present invention may further comprise the steps: with DNA template (a), and the step of using first primer, second primer relative and archaeal dna polymerase to carry out PCR with first primer; (b) will be connected with carrier of the present invention through the PCR product that step (a) obtains, obtain the step of cyclic DNA; (c) step that adopts the cyclic DNA obtain through step (b) host cell to be changeed shape; And (d) utilize such medicament to carry out medicament and select; Selection has the step of the transformant of cyclic DNA thus; Wherein said medicament is not have the drug resistance gene product of disappearance to chemical sproof medicament it illustrate, and said cyclic DNA forms through the PCR product is inserted carrier along desired orientation; Wherein, First primer has and aforementioned DNA complementary nucleotide sequence at 3 ' end side; And when the loss of activity drug resistance gene in the carrier of the present invention is when having lacked the gene of the nucleotide sequence that the C-terminal amino acid partly of drug resistance gene product is encoded; Said first primer has the nucleotide sequence that is lacked at 5 ' end side; When the loss of activity drug resistance gene in the carrier of the present invention is when having lacked the gene of the nucleotide sequence that the amino acid of the N-terminal of drug resistance gene product part is encoded, said first primer has at 5 ' end side and has removed 5 from the complementary strand sequence of the nucleotide sequence that lacked ' nucleotide sequence behind the terminal deoxythymidine.

Can design the nucleotide sequence or its complementary strand sequence that in the loss of activity drug resistance gene, are lacked in said first primer and the amino acid of the amino acid of requisite C-terminal part in resistance performance or N-terminal part is encoded like this; Make when the amplified production that will obtain through the PCR that uses this first primer is connected with cloning vector of the present invention, generated the sequence that the drug resistance gene product that does not have disappearance is encoded.For example, in the situation of the formed carrier of in using by the sequence number 5 of sequence table, putting down in writing in the sequence number 18 of the nucleotide sequence DNA of record and sequence table of nucleotide sequence DNA, can use at 5 ' end to contain 5 '-the nucleotide sequence primer of GGTAA-3 '.The aminoacid sequence of the aforementioned drug resistance gene product that does not have a disappearance or can be and natural type sequence different sequences to its nucleotide sequence of encoding is as long as gene product can be brought into play resistance.The inventor finds that the 290th amino acids with β-Nei Xiananmei is replaced into phenylalanine(Phe) and the varient (W290F) that obtains is the same with the wild-type β-Nei Xiananmei that resistance has been given play in the Ampicillin Trihydrate.According to this discovery; For example; The formed carrier of in will be by the sequence number 5 of sequence table, putting down in writing in the sequence number 18 of the nucleotide sequence DNA of record and sequence table of nucleotide sequence DNA is during as carrier, can use at 5 ' end to contain 5 '-the nucleotide sequence primer of TTTAA-3 ' or 5 '-TCTAA-3 '.In addition, the sequence of 3 ' end side of the 1st primer can have complementary and as the sufficiently long nucleotide sequence of PCR primer with template DNA.

Do not limit aforementioned the 2nd primer is special, but as long as it is an annealed primer in the elongation chain of the 1st primer, the 2nd primer can suitably design through known technology.Under the situation that is only obtaining the cyclic DNA that the PCR fragment inserts along specific direction, do not limit the 2nd primer is special, for example, can be the 2nd primer with the formed design of primers of a part of complementary nucleotide sequence with the elongation chain of the 1st primer.Under the situation of 2 kinds of cyclic DNAs that acquisition PCR product inserts along different directions respectively; The same with the 1st primer; Can design the 2nd primer, make it be included in nucleotide sequence or its complementary sequence that is lacked in the loss of activity drug resistance gene and the amino acid of the amino acid of indispensable C-terminal part in resistance performance or N-terminal part is encoded.

As aforementioned archaeal dna polymerase, can enumerate Pol I type archaeal dna polymerase, or contain the mixed type archaeal dna polymerase of above-mentioned polysaccharase as representational Taq DNA polymerase.For the dna fragmentation that utilizes after the widely used α type of Pol I type archaeal dna polymerase archaeal dna polymerase carries out pcr amplification; It is known because this archaeal dna polymerase has 3 ' exonuclease activity; Not having the outstanding dna fragmentation of dA at 3 ' end occupies the majority; But in PCR reaction back through handling as follows: add 3 ' end through Pol I type archaeal dna polymerase etc. to dA is outstanding, then can clone through the TA PCR cloning PCR.3 ' end as the dna fragmentation after carrying out pcr amplification through α type archaeal dna polymerase obtains the outstanding method of dA, and the 3 ' end that can enumerate the nucleotide sequence of the oriented Taq archaeal dna polymerase of apparatus or terminal deoxynucleotidyl transferase and so on adds the method that the active like this enzyme of base is handled.The dna fragmentation that use obtains through above-mentioned processing and the cloning process of carrier of the present invention also are one embodiment of the invention.

Use above-mentioned primer to and archaeal dna polymerase come the PCR reaction in the cloning process of embodiment of the present invention." the PCR Protocols " (the 2nd edition that the usage quantity of template DNA, primer, mg ion, 4 kinds of deoxyribonucleoside triphosphates, archaeal dna polymerase and the thermally denature/temperature and time of each technology of annealing/chain extending reaction and the cycle number of these technologies etc. can be write with reference to (for example) Baetlett JMS and StirlingD; Humana Press publishes, 2003) wait arbitrarily and set.

In being connected of PCR product in above-mentioned steps (b) and cloning vector of the present invention, can utilize dna ligase or commercially available connection test kit (ligation kit).Though not special qualification the among the present invention; But can give an example as dna ligase derives from the dna ligase of T4 phage, as connecting test kit can give an example DNA Ligation kit ver.1 (タカラバイォ Co., Ltd. system), DNA Ligation kit ver.2 (タカラバイォ Co., Ltd. system) and DNA Ligation Kit < Mighty Mix>(タカラバイォ Co., Ltd. system).In addition, when being connected with cloning vector of the present invention, the PCR product can be made with extra care, and also can the reaction solution behind the PCR directly is used for being connected with cloning vector of the present invention without refining.

As the host cell in the above-mentioned steps (c), among the present invention to its not special qualification, the colibacillus of can giving an example, withered grass bacterium, yeast and insect cell, wherein more preferably colibacillus.Conversion in the above-mentioned steps (c) adopts ordinary method to get final product, and for example can utilize the method or the electroporation that have used chemoreception attitude cell (chemical competent cell).

About employed selection medicament in the medicament of above-mentioned steps (d) is selected, using does not have the drug resistance gene product of disappearance that chemical sproof medicament it illustrate is got final product.For example, when the drug resistance gene product that does not have disappearance is β-Nei Xiananmei, can use the Ampicillin Trihydrate as selecting medicament.In addition, when cloning vector of the present invention has the 2nd drug resistance gene, can and with the gene product of the 2nd drug resistance gene to chemical sproof medicament it illustrate.

An embodiment of cloning process of the present invention is shown here, below.Though use the β-Nei Xiananmei gene to be illustrated here; But cloning process of the present invention is not limited to following object lesson; Can be right based on the suitable primer of the nucleotide sequence design of used drug resistance gene, clone through method identical or the conspicuous method of those skilled in the art thus with following method.

At first, preparation be used to increase right as the primer of the gene of clone's target.This primer centering; 5 ' end side of a primer adds base sequence GGTAA therein in advance, and this base sequence GGTAA makes the nucleotide sequence that the C-terminal amino acid Trp (W290) to the β-Nei Xiananmei gene encodes and the terminator codon in downstream thereof generate.Perhaps, can add base sequence TTTAA or the TCTA that makes that the terminator codon in amino acid Phe (F290) and downstream thereof generates.Use this primer and another primer on the other side, carry out PCR through Pol I type archaeal dna polymerase.Through doing like this, can obtain that any end at said amplified fragments is added with the sequence of GGTAA, TTTAA or TCTAA and so on and be added with the dna fragmentation of outstanding Desoxyadenosine at two 3 ' ends.Offer in the ligation with carrier of the present invention through the dna fragmentation that will obtain like this, can obtain only having inserted the cyclic DNA of dna fragmentation along desired orientation.

In the present invention, also can use 2 subtend primers that contain above-mentioned interpolation sequence at 5 ' end to implement PCR.In this case, can obtain to have added the sequence of GGTAA, TTTAA or TCTAA and so on and added the dna fragmentation of outstanding Desoxyadenosine at two 3 ' ends at two 5 ' ends.Offer in the ligation with carrier of the present invention through the dna fragmentation that will obtain like this, can obtain having inserted 2 kinds of cyclic DNAs of dna fragmentation along different directions.

Through aforementioned dna fragmentation being offered in the ligation with carrier of the present invention (it maintains the β-Nei Xiananmei gene that W290 has lacked) and being connected, make the carrier of ring-typeization.With the ligation of carrier of the present invention in, can just not make with extra care and use, the dna fragmentation after perhaps can using centrifugal post etc. refining above-mentioned dna fragmentation.The ring-type carrier that obtains like this through usefulness transforms the host, and through only selecting to have obtained the chemical sproof transformant in Ampicillin Trihydrate, can obtain having inserted along desired orientation the clone of nucleic acid fragment.

Therefore because cloning process of the present invention can obtain only to have inserted along desired orientation the cyclic DNA of dna fragmentation, in sequential analysis only, be useful to specific part.For example; With Bacteria Identification be categorized as purpose, when hundreds of bases of 5 ' side of 16S ribosomal RNA gene are carried out sequential analysis; Under the situation of the directivity that can not control dna fragmentation, the clone that the 3 ' regions that need not analyze is checked order is also carried out sequential analysis.Yet the carrier of the application of the invention can be eliminated the problems referred to above.Therefore, in aforementioned such distinguishing, can resolve rapidly and safely.In addition, had the nucleotide sequence that is rich in AT, but cloning process of the present invention also is useful in such dna fragmentation clone of being rich in AT by the DNA that widely used bisulf iotate-treated is crossed in the experimental embryology field.

Because cloning process of the present invention can obtain to carry out under the efficient TA clone at high positive colony; And can control the directivity of the dna fragmentation that is inserted; Therefore be in the purpose situation of cloning to insert the fragments sequence analysis; Also can prevent undisclosed clone is carried out the base sequence analysis, thereby can reduce the cost that base sequence is analyzed.

To can not limiting especially through the DNA (nucleic acid fragment) that carrier of the present invention is cloned.Can be the gene of peptide coding, gene or its fragment of encoding function property RNA (rRNA, miRNA etc.), can also be (for example) cis acting property base sequence key element (genetic expression control sequence or its candidates such as promotor, enhanser, silencer).In addition, also can the nucleotide sequence that function is not clear clone as the target nucleotide sequence and carry out the base sequence analysis.

(3) method of manufacture of carrier of the present invention

Do not limit the method for making carrier of the present invention is special, get final product so long as can make up the method for manufacture of carrier structure of the present invention.Do not limit following method capable of using to the carrier method of manufacture that has 1 outstanding dT residue at two 3 ' ends is also special: use can make the method for Restriction Enzyme preparation 3 ' dT protruding terminus of 1 base protruding terminus at 3 ' end of XcmI, MboII, HphI, MnlI, AhdI, Eam1105I etc.; And after the Restriction Enzyme of the smooth end through can preparing AluI, DpnI, RsaI, AfeI, DraI, EcoRV, HaeIII, PamCI etc. handles; Use has the active enzyme that adds base to 3 of the dna fragmentation of Taq archaeal dna polymerase or terminal deoxynucleotidyl transferase and so on ' end, prepares the method for 3 ' dT protruding terminus.Aforementioned limitations property enzyme can be selected according to the nucleotide sequence that the drug resistance gene that is carried in the carrier is encoded; But under the situation that can not find suitable Restriction Enzyme; Can be in the scope of the function that does not lose carrier of the present invention (for example; Aminoacid sequence is changed) change the base sequence of said nucleic acid, create Restriction Enzyme and cut off the position.

As carrier method of manufacture of the present invention, among the present invention it is not limited especially, but can enumerate the method that comprises the steps: the step of cutting off the circular double stranded DNA of the nucleotide sequence of record in the sequence number 19 with sequence table through Restriction Enzyme PmaCI; Thereby and through having two the 3 ' terminal step that obtains carrier of the present invention that the active enzyme of terminal deoxynucleotidyl transferase adds outstanding 1 deoxythymidine or uridine to cut double-stranded DNA.

Here, below show an embodiment of carrier method of manufacture of the present invention, it optionally obtains to have inserted the clone of nucleic acid fragment in the presence of the Ampicillin Trihydrate.

At first, in advance other drug resistance gene (second drug resistance gene) beyond the β-Nei Xiananmei gene is imported in the carrier that contains the β-Nei Xiananmei gene.During importing, can utilize the Restriction Enzyme site, also can utilize the nucleic acid amplification method of PCR and so on.Next make and terminal Trp of the β-Nei Xiananmei gene C that after importing, exists in the carrier and the corresponding excalation of terminator codon.As deletion method, can suitably utilize the inverse-PCR method.Do not limit this method is special, preferably use in aforementioned PCR its 5 ' end to be added with the right method of primer of Restriction Enzyme PmaCI recognition site.Cut off this amplified fragments through Restriction Enzyme PmaCI, next carry out afterwards the host being transformed, and aforementioned second drug resistance gene thing that serves as a mark is selected transformant from cyclisation through ligation.Through resulting transformant, select to maintain the transformant of the carrier that contains the β-Nei Xiananmei gene that C-terminal Trp lacked.To digest again by the carrier of this transformant preparation through Restriction Enzyme PmaCI, further add 1 outstanding dT residue to two 3 ' ends through archaeal dna polymerase or terminal deoxynucleotidyl transferase.Can prepare carrier of the present invention thus.

(4) test kit of the present invention

Test kit of the present invention contains carrier of the present invention and dna ligase.As aforementioned dna ligase, can enumerate the T4DNA ligase enzyme.In test kit of the present invention, can further contain the damping fluid of the composition of the ligation (ligation) that is suitable for carrying out through dna ligase.In addition, in test kit of the present invention, can contain the reagent (TaqDNA polysaccharase or terminal deoxynucleotidyl transferase etc.) that is useful on to the 3 ' terminal dA of interpolation that carries out the dna fragmentation that pcr amplification obtains through α type archaeal dna polymerase.

Do not limit test kit is special; The test kit that preferably carrier of the present invention and the combination of commercially available dna ligation kit is obtained, the test kit that for example obtains with DNA Ligation kit ver.1 (タカラバイォ Co., Ltd. system), DNA Ligation kit ver.2 (タカラバイォ Co., Ltd. system) or DNA Ligation Kit < Mighty Mix>combinations such as (タカラバイォ Co., Ltd. systems).

The test kit of the application of the invention, minimum work capable of using and time are cloned most unknown genes.Especially, the clone's test kit that can be used as the nucleic acid fragment that is rich in AT of nucleic acid fragment that bisulf iotate-treated crosses and so on.

Embodiment

Enumerate embodiment below and come the present invention is more specifically explained, but the present invention is not limited only to following embodiment.

In addition; In the operation of this specification sheets record; About elementary operations such as the preparation of plasmid, Restriction Enzyme digestion, carry out according to " molecular cloning: laboratory manual the 3rd edition (Molecular Cloning:A Laboratory Manual 3rd ed.) " middle method of putting down in writing of editors such as calendar year 2001 Cold Spring Harbor Laboratory distribution, J.Sambrook.

Embodiment 1

(1) structure of carrier

Carrier of the present invention makes up through following method.At first, through Restriction Enzyme Nde I and PvuII (both are by タカラバイォ Co., Ltd. system) pUC19 carrier (タカラバイォ Co., Ltd. system) is digested.Carrying out smooth endization afterwards handles with BAP.The dna fragmentation that obtains is like this made with extra care through ordinary method.Next synthetic dsdna, this double-stranded DNA have in the nucleotide sequence of record in the sequence number 5 of sequence table the nucleotide sequence from the 999th bit base to the 213 bit bases.Next, this double-stranded DNA is inserted among the pUC 19DNA that cuts off with Restriction Enzyme Nde I and Pvu II.With the DNA called after pUC19-Kana DNA that makes up like this.

With above-mentioned pUC19-Kana DNA is that template is carried out inverse PCR.At first, the AMP-F primer and the AMP-R primer that synthesize the base sequence of record in the sequence number 3,4 that has sequence table respectively.These primers are designed the 5 ' end side generation Restriction Enzyme PmaCI site that makes at these primers.Use the right PCR of this primer in following reaction solution is formed, to carry out.That is, contain 2 * PrimeStar Max Premix, 50 μ L, each 20pmol of above-mentioned primer and 2ng template DNA in the 100 μ L reaction solutions.In addition, the PCR condition is that 30 circulations as 1 circulation, are reacted in 98 ℃ of 10 seconds, (98 ℃ 10 seconds, 55 ℃ 5 seconds, 72 ℃ 40 seconds).PCR digests amplified production with Restriction Enzyme DpnI (precious biotechnology (Dalian) ltd system) after finishing.Afterwards, through Restriction Enzyme PmaCI (タカラバイォ Co., Ltd. system) digestion, and use DNA ligation kit (タカラバイォ Co., Ltd. system) to carry out from ligation.Reaction uses resulting cyclic DNA that e. coli jm109 (タカラバイォ Co., Ltd. system) is transformed after finishing.After resulting transformant is cultivated, prepare plasmid through ordinary method, and with this plasmid called after pUC19-bla Δ W290.This plasmid has the nucleotide sequence that β-Nei Xiananmei that C-terminal tryptophane (apart from N-terminal the 290th amino acids, corresponding base sequence is TCG) has been lacked is encoded.In fact the β-Nei Xiananmei of this C-terminal disappearance does not have the beta-lactam enzymic activity.

(2) affirmation of Ampicillin Trihydrate susceptibility

Plasmid pUC19-bla Δ W290 about preparation in above-mentioned (1) has confirmed Ampicillin Trihydrate susceptibility.That is, preparation contains totally 6 kinds of agarose LB flat boards of the Ampicillin Trihydrate that kantlex that concentration is 50 μ g/mL and concentration is respectively 0,20,40,60,80,100 μ g/mL.To be seeded in the above-mentioned flat board through the e. coli jm109 after plasmid pUC19-bla Δ W290 transforms, after cultivating a night under 37 ℃, calculate colony count.As a result, when Ampicillin Trihydrate concentration is 40 μ g/mL when above, do not observe colony growth.Thus, the intestinal bacteria after plasmid pUC19-bla Δ W290 transforms are that susceptibility this fact in Ampicillin Trihydrate obtains confirming.

(3) the T-carrierization of plasmid pUC19-bla Δ W290

Plasmid pUC19-bla Δ W290 to confirmed Ampicillin Trihydrate susceptibility through above-mentioned (2) carries out the T-carrierization.At first,, make with extra care after the plasmid pUC19-bla Δ W290 digestion with Restriction Enzyme PmaCI through removing sepharose.Next, add Taq archaeal dna polymerase and dTTP in the dna fragmentation after refining, make at 1 deoxythymidine residue of two 3 ' terminal interpolation.With the straight chain shape double chain DNA fragment called after pUC19-bla Δ W290-T DNA that so obtains.This double-stranded DNA is by being made up of the DNA that forms of nucleotide sequence of record in the sequence number 5 of sequence table and the DNA that is formed by the nucleotide sequence of putting down in writing in the sequence number 18 of sequence table.

Embodiment 2 inserts the PCR fragment of various chain lengths about the pUC19-bla Δ W290-T DNA of preparation among the embodiment 1 through the TA PCR cloning PCR, obtains efficient so that positive colony to be discussed.

(1) the segmental positive colony of 500bp PCR obtains the efficient discussion

Synthetic 500bp-F primer and the 500bp-R primer that has the nucleotide sequence of putting down in writing in the

sequence number

6,7 of sequence table respectively.Next, as template, use these primers to carry out PCR intestinal bacteria JM 109 genomic dnas.PCR carries out in following reaction solution is formed.That is, in 50 μ L reaction solutions, contain 2 * Ex Taq Premix (タカラバイォ Co., Ltd. system), 25 μ L, each 20pmol of above-mentioned primer and 50ng template DNA.In addition, the PCR condition be with 94 ℃ of 1 minutes, (98 ℃ 15 seconds, 59 ℃ 30 seconds, 72 ℃ 30 seconds) as 1 circulation, behind 30 circulating reactions, kept 5 minutes down at 72 ℃.Reacted amplified fragments is carried out sepharose to be reclaimed.

The 10 μ L reaction solutions of DNA Ligation kit ver.1 (タカラバイォ Co., Ltd. system) solution I that will contain pUC19-bla Δ W290-TDNA and the 5 μ L of the amplified fragments of 50ng after refining, 50ng kept 1 hour down at 16 ℃.As contrast, use pMD19-T carrier (precious biotechnology (Dalian) ltd system) to replace pUC 19-bla Δ W290-TDNA, in addition with above-mentioned the same enforcement reaction.Next, use each reaction solution after reaction is accomplished to come transformed into escherichia coli.After on the AIX flat board that resulting transformant is seeded into the Ampicillin Trihydrate that contains 100 μ g/mL concentration, cultivate an evening down at 37 ℃.To the clone's that forms thus a part, use primer that the base sequence by record in the sequence number 8 of sequence table forms and the primer that forms by the base sequence of putting down in writing in the sequence table sequence number 9 to carry out bacterium colony PCR.The PCR product that obtains is carried out agarose gel electrophoresis, and resulting result is shown in Fig. 1 and Fig. 2.

As shown in Figure 1, under the situation of using carrier of the present invention, confirmed in 32 clones, exist purpose to insert fragment among 32 clones (positive colony obtains efficient 100%).On the other hand, as shown in Figure 2, under the situation of the T-carrier that uses routine techniques, confirmed in 32 clones, exist purpose to insert fragment among 26 clones (positive colony obtains efficient 81%).Therefore, it is very high that the positive colony that has confirmed carrier of the present invention obtains efficient.

(2) the segmental positive colony of 1kbp PCR obtains the discussion of efficient

Synthetic 1K-F primer and the 1K-R primer that has the base sequence of putting down in writing in the

sequence number

10,11 of sequence table respectively.As template, use these primers to carry out PCR the e. coli jm109 genomic dna.The reaction solution of PCR is formed identical with above-mentioned (1) with the PCR condition.Reacted amplified fragments is carried out sepharose to be reclaimed.

The 10 μ L reaction solutions of DNA Ligation kit ver.1 (タカラバイォ Co., Ltd. system) solution I that will contain pUC19-bla Δ W290-TDNA and the 5 μ L of the amplified fragments of 50ng after refining, 50ng kept 1 hour down at 16 ℃.As contrast, replace implementing and above-mentioned the same reaction the pUC19-bla Δ W290-T DNA except using the pMD19-T carrier.Reaction uses this reaction solution to come transformed into escherichia coli after finishing.After being seeded into resulting transformant on the AIX flat board, cultivate a night down at 37 ℃.About the white clone of growth, use primer that forms by the base sequence of putting down in writing in the sequence number 8 of sequence table and the primer that forms by the base sequence of putting down in writing in the sequence number 9 of sequence table to carry out bacterium colony PCR.As a result, it is 100% that the positive colony of carrier of the present invention obtains efficient, and in contrast, in the situation of the T-of conventional art carrier, it is 90% that positive colony obtains efficient.Therefore confirmed that even be under the situation of 1kbp inserting fragment, it is also very high that the positive colony of carrier of the present invention obtains efficient.

(3) the segmental positive colony of 2kbp PCR obtains the discussion of efficient

Synthetic 2K-F primer and the 2K-R primer that has the base sequence of putting down in writing in the

sequence number

12,13 of sequence table respectively.As template, use these primers to carry out PCR the e. coli jm109 genomic dna.The reaction solution of PCR is formed identical with above-mentioned (1) with the PCR condition.Reacted amplified fragments is carried out sepharose to be reclaimed.

The 10 μ L reaction solutions of DNA Ligation kit ver.1 (タカラバイォ Co., Ltd. system) solution I that will contain pUC19-bla Δ W290-TDNA and the 5 μ L of the amplified fragments of 200ng after refining, 50ng kept 1 hour down at 16 ℃.As contrast, replace implementing and above-mentioned the same reaction the pUC19-bla Δ W290-T DNA except using the pMD19-T carrier.Reaction is used this reaction solution transformed into escherichia coli after finishing.After being seeded into resulting transformant on the AIX flat board, cultivate a night down at 37 ℃.About the white clone of growth, use primer that forms by the base sequence of putting down in writing in the sequence number 8 of sequence table and the primer that forms by the base sequence of putting down in writing in the sequence number 9 to carry out bacterium colony PCR.The result is shown in Fig. 3 and Fig. 4.

As shown in Figure 3, under the situation of using carrier of the present invention, confirmed in 15 clones, exist purpose to insert fragment among 13 clones (positive colony obtains efficient 87%).On the other hand, as shown in Figure 4, under the situation of the T-carrier that uses routine techniques, confirmed in 15 clones, exist purpose to insert fragment among 10 clones (positive colony obtains efficient 67%).Therefore confirmed that even be under the situation of 2kbp inserting fragment, it is still very high that the positive colony of carrier of the present invention obtains efficient.

Embodiment 3

Discuss about carrier of the present invention and the segmental relation of insertion.In the present embodiment, use the 500bp pcr amplified fragment and the 2kbp pcr amplified fragment of preparation in the foregoing description 2 (1) and embodiment 2 (3).The reaction solution of 6 kind of 10 μ L of preparation; It is carrier of the present invention and DNA Ligation kit ver.1 (the タカラバイォ Co., Ltd. system) solution I of 25ng, 50ng or 100ng that these reaction solutions contain with respect to the 500bp of 100ng or 2kbp amplified fragments respectively, and these solution were kept 1 hour down at 16 ℃.Identical among conversion and culture condition and the embodiment 2 (1).As a result, inserting under the segmental situation of 500bp PCR, can confirm the increase along with carrier concn, clone's number of acquisition is improved.On the other hand,, can confirm when the carrier amount is 50ng, obtain maximum clone's numbers inserting under the segmental situation of 2kbp PCR.

Embodiment 4

In the dna fragmentation that bisulf iotate-treated is crossed, GC content becomes and is rich in AT.In the present embodiment, discuss to the clone of the amplified fragments that is rich in AT, the amplified fragments of the said AT of being rich in obtains through widely used hydrosulphite PCR method in experimental embryology.

Synthesize primer that forms by the base sequence of putting down in writing in the sequence number 14 of sequence table and the primer that forms by the base sequence of putting down in writing in the sequence number 15.The human gene group DNA that bisulf iotate-treated is crossed uses these primers to carry out PCR as template.PCR carries out in following reaction solution is formed.That is, in the 100 μ L reaction solutions, contain 2 * Taq Hot Start Premix (タカラバイォ Co., Ltd. system), 25 μ L, each 20pmol of above-mentioned primer and 50ng template DNA.In addition, the PCR condition is that 30 circulations as 1 circulation, are reacted in 95 ℃ of 30 seconds, (95 ℃ 30 seconds, 53 ℃ 30 seconds, 72 ℃ 30 seconds).After the reaction, make with extra care amplified fragments through removing sepharose.In addition, this amplified fragments is 377bp, and through bisulf iotate-treated, GC content is reduced to 29% from 32%.The 10 μ L reaction solutions of DNALigation kit ver.1 (タカラバイォ Co., Ltd. system) solution I that will contain pUC19-bla Δ W290-T DNA and the 5 μ L of the amplified fragments of 100ng after refining, 50ng kept 1 hour down at 16 ℃.As contrast, replace implementing and above-mentioned the same reaction the pUC19-bla Δ W290-T DNA except using the pMD19-T carrier.Reaction is used this reaction solution transformed into escherichia coli after finishing.After being seeded into resulting transformant on the AIX flat board, cultivate a night down at 37 ℃.About the white clone of growth, use primer that forms by the base sequence of putting down in writing in the sequence number 8 of sequence table and the primer that forms by the base sequence of putting down in writing in the sequence number 9 to carry out bacterium colony PCR.As a result, it is 87.5% that the positive colony of carrier of the present invention obtains efficient, and in contrast, in the T-of conventional art carrier, it is 12.5% to 18.75% that positive colony obtains efficient.Therefore confirmed that even under the segmental situation of the insertion of being rich in AT, it is also very high that the positive colony of carrier of the present invention obtains efficient.

Embodiment 5

Synthetic 2k-R1 primer with base sequence of putting down in writing in the sequence number 16 of sequence table.With the e. coli jm109 genomic dna as template, with the 2k-F primer of embodiment 2 and above-mentioned 2k-R1 primer as primer to carrying out PCR.The reaction solution composition of PCR is identical with the condition of record among the embodiment 2 with reaction conditions.After the reaction, make with extra care amplified fragments through removing sepharose.This amplified fragments is 2kbp.Use amplified fragments and pUC19-bla Δ W290-T DNA after making with extra care, in following table 1, in the composition of the reaction solution I of record, under 16 ℃, carry out 1 hour ligation.As contrast, replace implementing and above-mentioned the same reaction the pUC19-bla Δ W290-T DNA except using pMD 19-T carrier.

Table 1

Reaction is used this reaction solution transformed into escherichia coli after finishing.After being seeded into resulting transformant on the AIX flat board, cultivate a night down at 37 ℃.About the white clone of growth, prepare plasmid through ordinary method after, the primer that will be formed by the nucleotide sequence of record in the sequence number 8 of sequence table checks order as sequencing primer.Situation about the situation of using carrier of the present invention and the traditional T-carrier that uses contrast obtains plasmid respectively from 10 bacterium colonies, confirm that it inserts segmental directivity.As a result, under the situation of using carrier of the present invention, insert fragment and be inserted in all plasmids along desired orientation.Relative with it, under the situation of the T carrier that uses conventional art, insert plasmid that fragment inserts along desired orientation and account for 60% of whole plasmids of being obtained.Therefore confirmed, insert segmental direction controlled aspect, compare with conventional art, carrier of the present invention has also obtained very high effect.

Embodiment 6

The end sequence that is inserted into the dna fragmentation in the carrier of the present invention further is discussed.

In multiple dna fragmentation being inserted into pUC19-bla Δ W290-T DNA to obtain the clone of anti-Ampicillin Trihydrate; And when the plasmid that maintains resulting clone is carried out sequence analysis, find that the 290th amino acids is that to be that the β-Nei Xiananmei of tryptophane is the same also given play to resistance to the Ampicillin Trihydrate for β-Nei Xiananmei and the 290th amino acids of phenylalanine(Phe).

Thereby, discuss about the segmental method of use of insertion that contains the base sequence that phenylalanine(Phe) is encoded at 5 ' end side.At first, synthetic sequence number by sequence table 20In the primer that forms of the base sequence of record and by sequence number 21The primer that the base sequence of middle record forms.Similarly, synthesized the sequence number that contains the base sequence that tryptophane is encoded at 5 ' end side by sequence table 22In the primer that forms of the base sequence of record and by sequence number 23The primer that the base sequence of middle record forms.As template, use these primers to carry out PCR e. coli jm109 pnca gene group DNA.PCR carries out in following reaction solution is formed.That is, in the 50 μ L reaction solutions, contain 2 * Ex Taq Premix (タカラバイォ Co., Ltd. system), 25 μ L, each 20pmol of above-mentioned primer and 50ng template DNA.In addition, the PCR condition be with 94 ℃ of 1 minutes, (98 ℃ 15 seconds, 59 ℃ 30 seconds, 72 ℃ 30 seconds) as 1 circulation, react 30 circulations after, kept 5 minutes down at 72 ℃.Reacted amplified fragments is carried out sepharose to be reclaimed.

The 10 μ L reaction solutions of DNA Ligation kit ver.1 (タカラバイォ Co., Ltd. system) solution I that will contain pUC19-bla Δ W290-TDNA and the 5 μ L of the amplified fragments of 50ng after refining, 50ng kept 0.5 hour down at 16 ℃.In addition, the reaction that the DNA Ligation Kit ver.1 that uses as reagent in use DNALigation kit < Mighty Mix>(タカラバイォ Co., Ltd. system) the replacement ligation is carried out is implemented according to the standard scheme of these goods.As contrast, replace implementing reaction same as described above the pUC19-bla Δ W290-T DNA in addition except using pMD 19-T carrier (precious biotechnology (Dalian) ltd system).Next, use each the reaction solution transformed into escherichia coli that reacts after finishing.After on the AIX flat board that resulting transformant is seeded into the Ampicillin Trihydrate of containing 100 μ g/mL concentration, cultivate a night down at 37 ℃.About the part of the bacterium colony of acquisition after cultivating, use primer that forms by the base sequence of putting down in writing in the sequence number 8 of sequence table and the primer that forms by the base sequence of putting down in writing in the sequence number 9 of sequence table to carry out bacterium colony PCR.PCR product to obtaining carries out agarose gel electrophoresis.

As a result, compare with using the situation when 5 ' end side contains the insertion fragment of the base sequence that tryptophane is encoded, use can obtain the bacterium colony more than 1.5 times in the segmental method of insertion that 5 ' end side contains the base sequence that phenylalanine(Phe) is encoded.In addition; Compare with the situation of use when 5 ' end side contains the insertion fragment of the base sequence that tryptophane is encoded; With carrier of the present invention, when 5 ' end side contains the insertion fragment of the base sequence of phenylalanine(Phe) coding and Ligation Kit < Mighty Mix>combination, can obtain the bacterium colony more than 3 times.In addition, for the clone who obtains through the reaction of using pUC19-bla Δ W290-T DNA, when detecting its positive colony acquisition efficient, positive colony obtains efficient and is 100% under arbitrary condition.On the other hand, under situation about using as the pMD19-T carrier that contrasts, though the number of the positive bacterium colony (white colony) that obtains is more, it is 33% to 50% that positive colony obtains efficient.Therefore can know that will have the method that the dna fragmentation to the base sequence of phenylalanine(Phe) coding is inserted in the carrier of the present invention at 5 ' end side is a preferred embodiment of the inventive method.

Embodiment 7

In embodiment 6, it is preferred can confirming to use the insertion fragment that all is added with the base sequence of phenylalanine(Phe) coding at two 5 ' end sides.In the present embodiment, be added with for 5 ' end side the insertion fragment of the base sequence of phenylalanine(Phe) coding is discussed in a side.That is, synthesize the sequence number that contains the base sequence that phenylalanine(Phe) is encoded at 5 ' end side by sequence table 20In the primer that forms of the base sequence of record and do not have the sequence number that adds sequence at 5 ' end side by sequence table 24The primer that the base sequence of middle record forms.In addition, synthesize the sequence number that contains the base sequence that tryptophane is encoded at 5 ' end side by sequence table 22The primer that the base sequence of middle record forms.E. coli jm109 pnca gene group DNA as template, is used the sequence number by sequence table 20In the primer that forms of the base sequence of record and by the sequence number of sequence table 24The primer that the base sequence of middle record forms carries out PCR and uses the sequence number by sequence table 23In the primer that forms of the base sequence of record and by the sequence number of sequence table 24The primer that the base sequence of middle record forms carries out PCR.PCR carries out in following reaction solution is formed.That is, in the 50 μ L reaction solutions, contain 2 * Ex Taq Premix (タカラバイォ Co., Ltd. system), 25 μ L, each 20pmol of above-mentioned primer and 50ng template DNA.In addition, the PCR condition be with 94 ℃ of 1 minutes, (98 ℃ 15 seconds, 59 ℃ 30 seconds, 72 ℃ 30 seconds) as 1 circulation, react 30 circulations after, kept 5 minutes down at 72 ℃.Reacted amplified fragments is carried out sepharose to be reclaimed.

The 10 μ L reaction solutions of DNA Ligation kit < Mighty Mix>(タカラバイォ Co., Ltd. system) that will contain pUC 19-bla Δ W290-TDNA and the 5 μ L of the amplified fragments of 50ng after refining, 50ng kept 0.5 hour down at 16 ℃.As contrast, replace implementing reaction and cultivation same as described above the pUC 19-bla Δ W290-T DNA except using pMD 19-T carrier (precious biotechnology (Dalian) ltd system).For the part of the bacterium colony that obtains after the cultivation, use primer that forms by the base sequence of putting down in writing in the sequence number 8 of sequence table and the primer that forms by the base sequence of putting down in writing in the sequence number 9 of sequence table to carry out bacterium colony PCR.PCR product to obtaining carries out agarose gel electrophoresis.

The result; Used 5 ' end side in a side have the segmental situation of insertion of the base sequence of tryptophane coding in and used 5 ' end side in a side to have in the segmental situation of insertion to the base sequence of phenylalanine(Phe) coding; The colony count that obtains is roughly the same, and the positive colony pick-up rate is 100% in arbitrary situation.In situation about using as the pMD19-T carrier that contrasts, though the quantity of the positive bacterium colony (white colony) that obtains is more, it is 40% that positive colony obtains efficient.Therefore can confirm that will have the method that the dna fragmentation to the sequence of phenylalanine(Phe) coding is inserted in the carrier of the present invention at 5 ' end side of a side is a preferred embodiment of the inventive method.

Though the present invention is specified with reference to specific embodiment, it should be apparent to those skilled in the art that under the situation that does not break away from the spirit and scope of the present invention, can carry out various changes and modification.

The application is based on 3 September 2010 filed Japanese Patent Application (Patent Application No. 2010-197380) and December 24, 2010 filed Japanese Patent Application (Patent Application No. 2010-286834), the contents of the above-mentioned patent applications incorporated by reference this article.

Utilizability in the industry

The carrier of the application of the invention can be selected the clone who is inserted with goal gene along desired orientation with easy operation.Therefore, the present invention especially can be used for the genetically engineered field, uses in life science and the medical field etc.

Sequence list text none

SEQ ID NO:1; Oligonucleolide primers as the Kana-F design

SEQ ID NO:2; Oligonucleolide primers as the Kana-R design

SEQ ID NO:3; Oligonucleolide primers as the AMP-F design

SEQ ID NO:4; Oligonucleolide primers as the AMP-R design

SEQ ID NO:5; The positive chain-ordering of pUC19-bla Δ W290-T

SEQ ID NO:6; Oligonucleolide primers as the 500bp-F design

SEQ ID NO:7; Oligonucleolide primers as the 500bp-R design

SEQ ID NO:8; Be used for the Oligonucleolide primers that bacterium colony PCR is designed

SEQ ID NO:9; Be used for the Oligonucleolide primers that bacterium colony PCR is designed

SEQ ID NO:10; Oligonucleolide primers as the 1k-F design

SEQ ID NO:11; Oligonucleolide primers as the 1k-R design

SEQ ID NO:12; Oligonucleolide primers as the 2k-F design

SEQ ID NO:13; Oligonucleolide primers as the 2k-R design

SEQ ID NO:14; The Oligonucleolide primers of crossing for the bisulf iotate-treated that increases that the human gene group DNA designed

SEQ ID NO:15; The Oligonucleolide primers of crossing for the bisulf iotate-treated that increases that the human gene group DNA designed

SEQ ID NO:16; Oligonucleolide primers as the 2k-R1 design

SEQ ID NO:17; Gene to the inactivation mutant code of β-Nei Xiananmei

SEQ ID NO:18; The minus strand sequence of pUC19-bla Δ W290-T

SEQ ID NO:19; The nucleotide sequence of plasmid pUC19-bla Δ W290

SEQ ID NO:20; That is designed contains the Oligonucleolide primers to phenylalanine(Phe) coded DNA sequence at 5 ' end regions

SEQ ID NO:21; That is designed contains the Oligonucleolide primers to phenylalanine(Phe) coded DNA sequence at 5 ' end regions

SEQ ID NO:22; That is designed contains the Oligonucleolide primers to tryptophane coded DNA sequence at 5 ' end regions

SEQ ID NO:23; That is designed contains the Oligonucleolide primers to tryptophane coded DNA sequence at 5 ' end regions

SEQ ID NO:24; In order to increase from the Oligonucleolide primers that dna sequence dna designed of bacillus coli gene group DNA

Claims

1. carrier; It is the cloning vector that is formed by straight chain shape double-stranded DNA; Said straight chain shape double-stranded DNA has outstanding 1 deoxythymidine or uridine residue at two 3 ' ends; This carrier is characterised in that; It maintains the nucleic acid that the partial sequence of drug resistance gene product is encoded, and said nucleic acid is the loss of activity drug resistance gene that has lacked such nucleotide sequence, and this nucleotide sequence is encoded to the amino acid of indispensable C-terminal part in the resistance performance of said drug resistance gene product or the amino acid of N-terminal part; In addition; Said nucleic acid is configured in the said double-stranded DNA end of any one, makes when the insertion dna fragmentation that contains the nucleotide sequence that is lacked in the said loss of activity drug resistance gene at terminal portions wherein is connected to carrier, has constituted the nucleic acid that the drug resistance gene product that not have to lack is encoded.

2. the described carrier of claim 1, wherein, said loss of activity drug resistance gene is the loss of activity drug resistance gene that has lacked such nucleotide sequence, this nucleotide sequence is encoded to the amino acid of the C-terminal part of drug resistance gene product.

3. the described carrier of claim 2, wherein, said loss of activity drug resistance gene is formed by the nucleotide sequence that the aminoacid sequence that has lacked the amino acid whose β-Nei Xiananmei of C-terminal is encoded.

4. the described carrier of claim 3, wherein, said loss of activity drug resistance gene is formed by the nucleotide sequence that the aminoacid sequence of record in the sequence number 17 of sequence table is encoded.

5. the described carrier of claim 1 also comprises the 2nd drug resistance gene, and said the 2nd drug resistance gene illustrates the resistance to medicament different with aforementioned drug resistance gene.

6. the described carrier of claim 1, DNA that it is formed by the nucleotide sequence by record in the sequence number 5 of sequence table and the DNA that is formed by the nucleotide sequence of putting down in writing in the sequence number 18 of sequence table form.

7. the method for a cloned DNA may further comprise the steps (a)～(d):

(a) be template with DNA, the step of using first primer, second primer relative and archaeal dna polymerase to carry out PCR with first primer;

Each described carrier is connected in the PCR product that (b) will obtain through step (a) and the claim 1～6, obtains the step of cyclic DNA;

(c) step that adopts the cyclic DNA obtain through step (b) that host cell is transformed; And

(d) utilize the drug resistance gene product that does not have disappearance that chemical sproof medicament it illustrate is carried out medicament and select, selection has the step of the transformant of cyclic DNA thus, and wherein said cyclic DNA forms through the PCR product is inserted carrier along desired orientation;

Wherein, Said first primer has and aforementioned DNA complementary nucleotide sequence at 3 ' end side; And when the loss of activity drug resistance gene in the carrier that right requires to put down in writing in 1～6 is when having lacked the gene of the nucleotide sequence that the C-terminal amino acid partly of drug resistance gene product is encoded; Said first primer has the nucleotide sequence that is lacked at 5 ' end side; When right requires loss of activity drug resistance gene in the carrier of record in 1～6 is when having lacked the gene of the nucleotide sequence that the amino acid of the N-terminal part of drug resistance gene product is encoded, and said first primer has at 5 ' end side and removed 5 from the complementary strand sequence of the nucleotide sequence that lacked ' nucleotide sequence behind the terminal deoxythymidine.

8. the described cloning process of claim 7, wherein the archaeal dna polymerase in the step (a) has the activity of adding 1 Desoxyadenosine base at 3 of reaction product ' end with the dependent mode of non-template.

9. the described cloning process of claim 7 wherein, also is included in the step that 3 of reaction product ' end adds 1 Desoxyadenosine base in step (a).

10. the method for manufacture of a carrier comprises the following steps: to cut off through Restriction Enzyme PmaCI the step of the circular double stranded DNA of the nucleotide sequence of record in the sequence number 19 with sequence table; And through having the active enzyme of terminal deoxynucleotidyl transferase, thereby two 3 of the double-stranded DNA after cut-out ' terminally add the step that outstanding 1 deoxythymidine or uridine residue obtain the carrier described in the claim 1.

11. a test kit, it uses test kit for the clone, contains any described carrier and dna ligase in the claim 1～6.