JPH03501331A - Phage vector λSK9 for generation of genomic library - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Genomic library preparation Phage vector λSK9 for Technical field The present invention relates to biotechnology, and more particularly to new technologies for the production of genomic libraries. The present invention relates to the specific phage vector λSK9.

Conventional technology The production of genomic libraries, including those intended for use in biotechnology, is the most important first step in the molecular biological study of gene structure and function. Ru.

In recent years, phages of the EMBL series have been widely used for the above purposes. (Prischauf A, M, Lehrach H, Murray N, .

J, Mo1. Biol, 1983, Vol, 170.827-854>.

These phages (λEMBL4.λEMBL3.λEMBL3A) are EcoR I and Bam I-Sl-5au3A (I> Genome according to the recognition site of the restriction enzyme used for the production of libraries (the "classical" method).

However, these vectors have a number of drawbacks, the main one being the polyphosphorus The existence of recognition sites for only three types of restriction enzymes in the car, and the mapping of insertion sites Difficulties encountered in insert segment-specific labeling without special recloning Impossible, recloning itself is troublesome.

These vectors belong to the classical and commonly used vectors, and are also part of the cDNA special table 13-50 1331(2) Plasmids for further manipulation (restriction mapping, sequencing) by insertion vector or M13 phage vector. stomach.

A more complete vector λCh40 is known in the art (Dunn 1. S,, Blattner, F, R,, Nucl, Acad, Res,, 198 7, νo1.15.2677-2798). In this vector, 16 types Cloning can be performed for many types of restriction enzymes, and all known methods A gene library can be created by this method. The main district of this vector Distinguishable features include approximately 80 repeat copies with a length of 235 base pairs In the structure of the insert. This repeating unit contains the 1ac-operator as well as the restriction enzyme It contains the recognition sites for the elementary Xamm and NaeI. 1ca-operator's Due to the presence of phage λCh40 in the presence of X-ga1 dye, the corresponding E. coli It becomes possible to give the plant a blue plaque. By following the first (classical) method The gene library in vector Ch40 is formed as follows. Ch 40 DNA was hydrolyzed using restriction enzymes Bam I and Nac I, and A repeating unit with a length of 235 base pairs is defined as an “arm” by the properties tool. It is removed by precipitation. Next, these “arms” are Mho I (Sa, u 3 ligated with a fragment of genomic DMA hydrolyzed by AI). resulting library The percentage of recombinants in the sample can be easily determined by color identification. recombinant gives white plaques, while the first phage gives blue plaques. This base The main advantage of this vector is that it uses E. coli rec A-strain cells as the host. This gives the possibility of creating a library of On the other hand, for rec A+ stock It is difficult to predict the outcome of manipulation of λCh40 in This is because the 80 iterations that exist lead to undesired results. In addition, complete DNA This vector requires 80 times the amount of the expensive restriction enzyme Nac I for cleavage. Operation with a vector is not economically convenient.

Furthermore, the vectors λDASH and λFIX are known in the art (r (Published by ``Stratesene'', Inc., USA). Vector λDASH is “classical” It is intended for generating gene libraries by a method of λEMBL The main advantage of this vector over 3 is the ability to clone and excise DNA inserts. additional sites for extrusion, as well as the enzyme RNA-polymerase A promoter that allows the segment of the DNA insert directly adjacent to the membrane to be labeled. The existence of T3 and T7.

These labeled segments are located at overlapping insertions (“along the chromosome”). can be used as a sample for the search for phages containing Wear. However, these advantages are not obvious. Because typical libraries Additional restriction enzyme recognition sites in the polylinker are of particular importance to provide On the other hand, the presence of large numbers of repeating units in the DNA of mammalian plants and other organisms , there is a high probability that these repeat units are labeled from promoters T3 and T7. This is because it leads to Labeled repeat units “walk along the chromosome” It is clear that it is not suitable for use in

Vector λFIX is used to generate gene libraries using site-specific completion of construction. intended for However, in this case, the hydrolysis of phage DNA is This is done using the restriction enzyme XhoI, which gives similar cohesive ends, instead of using . This is related to the following fact. That is, phage FIX (and DAS) I) is 5 It has a pi-phenotype. That is, this is a cell that is lysogenic with respect to phage P2. on the other hand, hydrolysis with Sal I abolishes this phenotype. breaking and leading to other undesirable consequences. Similar to λD A S H, this vector - has additional recognition sites for restriction enzymes in the polylinker compared to λEMBL3 as well as Contains promoters T3 and T7. λFIX and λDASH are A disadvantage is that the presence of recombinants in a library cannot be determined quickly.

Disclosure of invention The main purpose of the present invention is to provide a new phage vector, and The vector should be selected against non-recombinant phages and the possibility of co-infection of the library. to eliminate the potential for the terminal site of the DNA segment to be cloned. Introducing the desired marker and transferring the insert into a plasmid or single-stranded form and make it possible.

For this purpose, we provided the phage vector λSK9 for the production of a genomic library. According to the present invention, the vector has 47.7 t, n, has size p and has size 20.3 t, n, f), 77 One diλEMBL3-7 arm Co5-5al l-DNA fragment: 3.8 Sal I-Bgl of phasmid 5K2A with size t, n, p ll-DNA fragment (BcoR1 site is destroyed upon completion of the production method and the fragment Asmid 5K2A was produced from vector SK1g, which contains the DNA of phage λ. fragment, produced from phage M13 and Nismid J)) Ic79); Phage λHMBL30Ban+ with sizes of 13,7 t, n, p, 1-Ram l-DNA fragment; Ram 1 of the plasmid 5K2A having the sizes of 0, 7, t, n, p, -BgI n-DNA fragment (Sal 1 site has been removed upon completion of the method) ): 9.2 Phage λEMBL3-light arm with size t, n, P Sal1-Co5-DNA fragment; recognition of restriction enzymes Bam I and EcoRI The capacity of the vector consists of: a site that allows insertion of foreign DNA according to the R position; 4.4-19, Ot, n, P, and the spectrum of the E. coli strain that is the host of the vector. Kutle is LE392=F', had R514 (rs+-, mh-)e 5upE4 4. sup F1a.

lac Yl 02 (lca I ZY)6. gal K2. gal T2 2.　met　Bl.

trp R55; J, M, 109=rec Al, Δlac pro, end AI, g yr　AB6. thi-1゜hsd R17, sup E44. rel A l, F’, tra 036゜pro AB”, lac l’Z, 6M1 5: Q359=hsd Rm-, had L+”, sup F, φ80. P2O; All-Union Collection of Industrial Microorganisms of the ll5sR Research Institute for genetics and industrial microorganisms ・Bleeding (All-IJnion Co11ection of In industrial Microorganisms of the USSRRe 5earch In5 posture for Genetics and In PHN (industrial Microorganism Breeding) 1687 and was deposited on July 21, 1989.

The vectors presented here are constructed using standard methods from a representative genomic library. intended for fabrication.

The vector proposed here makes it possible to select against non-recombinant phages, This property improves library quality and saves time and materials; the vector It makes it possible to determine the presence of recombinant phages according to their phenotypic characteristics, thereby provides the possibility to constantly monitor the quality of the library: A radioactive marker or any other marker is placed at the end of the DNA segment to be allows for the introduction of markers, which are useful for subsequent manipulation of the segment. This is very important because it allows you to get the pieces you need without incurring additional steps. This allows research to reduce the time needed to obtain final results. The vector can be quickly and effectively flushed if needed. It is possible to convert from the phage form to the plasmid form without special additional cloning. The vector can be prepared in single-stranded form for determination of primary DNA structure by Sanger method. can be converted to

Some individual of the above properties are encountered in commercially available vectors of the prior art. However, their combination is essential only in the vector proposed here. be.

Brief description of the drawing The invention will now be described by a detailed description of exemplary embodiments thereof and with reference to the accompanying drawings. do. In these figures, Figure 1 (a, b) shows diphasmid. id) Schematically shows 5K18' production: Figure 2 schematically shows the preparation of diphasmid 5K1B; Figure 3 shows the preparation of phasmid gS. Schematically shows the construction of vector λgES7 from R: Figure 4 schematically shows the construction of phage vector λ5K90; The production of a gene library is schematically shown.

BEST MODE FOR CARRYING OUT THE INVENTION The phage vector λSK9 provided herein was constructed according to the scheme below. Ru. First, vector 5K18 is constructed (Fig. 1 a, b, and Fig. 2).

The size of phage M13a + p18 [1.86 t, n, P, from lNA] The fragment containing the protein was cut out using restriction enzymes Bal I and Bgl II, and Purify by electrophoresis in spot agarose. This fragment is a segment of phage M13. Genetics of operator, promoter, and lactose operon in addition to Mentoori progeny 1aci and Iacz, polylinker sequences, and in phage of the mp series. Contains a segment complementary to a standard oligonucleotide primer for sequence It consists of At the same time, Nismid pHc79 was treated with restriction enzymes EcoRI and SalI. more hydrolyzed. Next, the Klenow cleavage of DNA polymerase I from E. coli was performed. cohesive ends of the purified fragment containing the ori (segment of phage M13) The sticky ends of the resulting fragments of , and Nismid pHC79 were blunted and ligation was performed. cormorant. Introducing the ligated DNA into E. coli strain JM 109 using standard methods, DNA was isolated according to standard methods and restriction enzymes EcoR1, PstI, Analyze using Hindm, Baall, Bglm, and Sall. Ru. The resulting diphasmid SK 1 is used for the next operation. reduce size SK1 was hydrolyzed with the restriction enzyme old ndII [and Bgl II and then purify the fragment containing the gene for ampicillin stability. simultaneous Next, the DNA of phage M13mp13 was treated with the same restriction enzyme, and low molecular fragments were isolated. both fragments were ligated, and cells of E. coli strain JM109 were injected into the lysate thus obtained. The transformed cells were treated with ampicillin, X-ga 1 and medium containing IPTG is inoculated, DNA is isolated from them, and then the DNA restriction enzyme EcoR1, Pstl.

)1indIII, Bao+L BglI [and SalI cleaved and analyze it. The phasmid SK2 is isolated and used in the next step.

The DNA of Nismid pHC79 was developed with the restriction enzyme Baln, and COS Isolate the fragment containing the segment. Hydrolyze the SK2 DNA and c Ligate with the fragment containing the os segment. Next, cells of E. coli JM109 strain were prepared. The resulting solution was hydrolyzed and these cells were treated with ampicillin. Inoculate the medium. Reply from grown colonies onto nitrocellulose filters. with the Bgl fragment of Nismid pHc79, which creates a mosquito and contains the segment COS. Hybridization with 32P eJ by nick translation zation. Uncover cells from colonies that hybridize with COS fragments. Grow in medium containing picillin and isolate DNA, which is treated with the restriction enzyme Ec oRI, Pst I, Hind m, Bam I, BglII.

Cleaved with Sal I and analyzed. One of the isolated diphasmids, Diphasmid SK6 is used in the next step.

Diphasmid SK6 was hydrolyzed with the restriction enzyme EcoRI, and the cohesive ends were converted to dNT. complemented with the Klenow fragment in the presence of R and purified the linear diphasmid. and ligate, and the resulting solution was used to form cells of E. coli JM109. Transformed and inoculated into a medium containing ampicillin X-ga 1 and IPTG. do. Cells were grown from colonies in medium containing ampicillin and Isolate asmid DNA. The DNA was digested with the restriction enzyme EcoRI.

By Pst I, Hind m, Bam I, BglII, Sal I The sample is then cleaved and analyzed. Diphasmid 5K18 from analyzed DNA ’ is isolated. This was hydrolyzed with restriction enzyme Acc I, and the polylinker The sticky end of the Acc I site that exists outside of the Fill with Klenow fragment below. This preparation was added to phage T4 D in buffer. Treat with NA IJ gauze, and then use the resulting solution to isolate E. coli JM109 cells. transform cells containing ampicillin, X-ga1 and IPTG. Inoculate the medium. Cells from this colony were grown in medium containing ampicillin. , and isolate the DNA, which is cleaved with Acc I and analyzed. Only Diphasmid 5K18 containing the Acc1 site of is isolated.

Next, the DNA of diphasmid 5K18 was hydrolyzed with restriction enzyme Bgl II. , self-ligation is performed, and the ligated DNA is used to infect E. coli strain JM109 cells. and inoculate the cells into medium containing ampicillin. ampicilli Cells are grown from colonies grown on medium containing DNA, and DNA is extracted from them. was isolated by alkaline lysis, and the DNA was purified using EcoRI, BamI (I).

Cleaved with SalI, BglII and analyzed. These analyzes From these phasmids, phasmid 5K2A is isolated.

Phasmid 5K2A contains restriction enzymes EcoRI and It differs from phasmid SK2 in that it lacks the recognition site for Acc. Next, the fas The recognition site of the restriction enzyme Sal I in mid-5KZA is removed. for this purpose , phasmid 5K2A DNA was hydrolyzed with restriction enzyme Sal I, and The sticky ends are blunted with the Klenow fragment of DNA polymerase I. Next The ligated DNA is self-ligated into E. coli JM109 cells. child DNA was isolated from these colonies by standard methods and treated with the restriction enzymes EcoR1,B. Analyze by am Hl, Sal I. BcoRI.

Contains a Bam I restriction enzyme recognition site but also a Sat I restriction enzyme recognition site isolate the phasmid SK2ΔS that does not.

Next, the recognition site of the restriction enzyme EcoRI in phasmid SK2ΔS is removed. child To do this, the DNA of phasmid SK2ΔS was hydrated with the restriction enzyme EcoRI. The sticky ends are then blunted with Klenow fragment. After this, the DNA Perform self-concatenation. The ligated DNA is cleaved using the restriction enzyme EcoRI, Then, it is introduced into E. coli JM109 cells. D from colonies according to standard methods. Isolate the NA and use the restriction enzymes EcoRI, Baml and SalI and analyze it. Contains Bam I restriction enzyme recognition site but EcoRI and S A phasmid SK2ΔSR that does not contain an alI restriction enzyme recognition site is isolated.

After this, recombinant phasmid gSR is produced. Restriction enzyme DNA of phage λgt Hydrolyzes and inactivates the enzyme. Next, phage λ Anneal the cohesive ends and center with a size of approximately 6.5 t,n,p. Purify the Bam I fragment (Preparation A). DNA of the phasmid SK2ΔSR was hydrolyzed with the restriction enzyme Bam I and after inactivation of the enzyme was prepared as Preparation A. Link. The ligated DNA was introduced into E. coli JM109 cells, and the amplicon Contains syringe and inoculates into agar. Nitrocellulose filaments from proliferating shita colonies Take a replica on the router and mark it with 32P by nick translation. Hybridization with Preparation A containing the identified DNA is performed. , Preparation A cells from colonies that hybridize with the plasmid DNA A is isolated by standard methods. The restriction enzyme EcoRI of the DNA thus obtained , Bam I, Sal I showed that the phasmid of segment λgt■ Confirm cloning in SK2ΔSR. The obtained Fasmid gSR will be used in the next stage. Use on the floor. Furthermore, phage vector λgES70 is constructed (Fig. 3).

To do this, the DNA of plasmid gSR was hydrolyzed with the restriction enzyme Bgl II. and inactivate this enzyme.

The DNA of phage λEMBL3 was hydrolyzed with restriction enzyme Bam I, and this Inactivates enzymes. Both DNA preparations are mixed equimolarly and ligated. Linking The resulting DNA is packaged in-test into phage protein and the resulting DNA is The phage particles were used to plot cells of E. coli strain LE392 containing 0.1% X-gal. inoculate the animals. Propagate phage from 10 blue plaques and isolate the DNA. Release and restriction enzyme EcoRI, Bam 1. Analyze by Sal.

Phage λgES7 is isolated and used in the next step.

The DNA of phage λEMBL3 was hydrolyzed with the restriction enzyme Bam I, and approximately 1 Purify the insert with a size of 3.7 kbpO (Preparation B). Phage λ The gES7 DNA was hydrolyzed with the restriction enzyme BamI, and the "arms" were purified. (Preparation C). Preparations B and C are mixed equimolarly and combined. connected The obtained DNA is packaged in vitro into the protein of phage λ, and this The phage particles thus obtained are inoculated into a plot of cells of E. coli strain LH392. . Phage were formed from six plaques, D and NA were isolated, and restriction enzyme Analyze using elementary EcoRI, Bam I and Sal I. It is called λSK9. One of the phages is isolated. This structure is as shown in FIG.

The phage vector λSK9 proposed here is a “classical” vector as shown in Figure 5. ” method is convenient for obtaining representative genomic libraries.

The DNA of phage vector λSK9 was treated with a 10-fold excess of restriction enzymes EcoR1 and Ba. Hydrolyzed by mI. Next, the part where DNA is remain inside. After dissolving the DNA precipitate, the λSK9 with cohesive end Bam I was isolated. Terminal fragments (“arms”) and cohesive ends) l! An intermediate fragment with coRI is obtained.

Partial hydrolysis of genomic DNA with restriction enzyme 5au3AI (or MboI) and isolate fragments with sizes of 15-20t, n, p (gel electrolysis). (by electrophoresis or centrifugation). Phage DNA from which these fragments were treated as described above A, and the ligated DNA is packaged into the protein of phage λ in vitro. , and the resulting phage particles are used to infect E. coli cells. In this case general Approximately 70% of the phages propagated in this study are recombinant.

Recombinant phages were conserved in the λSK9 selection and the initial non-recombinant λSK 9 is Phage P2 (spi phenotype) lysogen against Q359,8M539 strain cannot replicate on E. coli cells that are sexually active.

The DNA insert in λSK9 can be easily transferred to plasmid form. this The principle used in this case is different from that used with λZAP. Recombinant λSK9 The DNA of Water is cleaved and the enzyme is inactivated by three freeze/thaw cycles.

Both restriction enzymes cleave nucleophilic DNA at sufficiently low frequencies (Sal I on average has one recognition site per about 50 kbp, and C1a1 has about 2 recognition sites on average. 1 recognition site per 0 kbp).

The DNA is then diluted and self-ligated. The ligated DNA is combined with E. coli. transduced into Tens cells and inoculated onto agar agar containing ampicillin. increase The grown colonies have the insert in the form of a plasmid, which contains the phage λ. Contains no nucleotide sequence or a small portion thereof (in the case of C1aI) ). Transfer to plasmid form is rapid (3-4 hours) and effective (0.1 g recombinant DNA (giving several thousand colonies) is realized.

Other routes of transfer to plasmid form also exist for λSK9 and its recombinants. do. In other words, the direct introduction of phages into E. coli cells that are resistant to cytolytic replication of phage λ. infection, but this route is not convenient and for special experiments It is useful.

In superinfection of E. coli cells containing the plasmid with phage M13, the plasmid Sequence the insert DNA according to the well-known method of Sanger. It is packaged into phage proteins in single-chain form, which is convenient for carrying out this process.

The capacity of λSK9 is up to 19 kbp, which is a typical genomic library. is quite sufficient to obtain. Because the average insert usually consists of 15 kbp It is et al.

Therefore, the vector λSK9 proposed here is representative by the "classical" method. can be used to create a unique genomic library. proposed here The main advantages of this vector are:

Obtain gene libraries without the need for prepurification of phage “arms” ・Be able to select against non-recombinant phage; ・ Rapid, effective and controlled conversion of phage λ to “arm”-free plasmid form ・Determination of primary structure of DNA by Sanget's method It is possible to transition to a single-chain form for: ・It is guaranteed that co-infection of the library in λSK9 will not result in a sudden infection. ; - 5'-3'- and and other segments.

For a better understanding of the present invention, the construction of vectors and their use are proposed herein. Illustrative examples are described below.

Example 1゜ First, diphasmid SK1g is produced. The fabrication scheme is shown in Figures 1 and 2. Shown below. Restriction enzymes Bal I and B from DNA5ag of phage M13mp18 Size of 1, 86 t, n, P using gl II (10 activity units each) The fragment with the do. This fragment contains the segment ori of phage M13 plus an additional operator. promoter, lactose operon genes 1ac i and 1ac Z Standards for s polylinker sequences and sequences in mp series phages a segment complementary to a target oligonucleotide primer. Next, Sumid pHpHC79(2) was combined with restriction enzymes EcoRI and 5ail(2) under standard conditions. 20 activity units each). In the second step, DNA from E. coli With Klenow fragment of polymerase I, 20mM Tyis-HCj! ( pH 7,4), 5mM MgC12, 10mM 2-mercaptoethanol, 50 All four species in buffer 5I containing mM NaCl 1°5/rrLlDNA Phage M1 in the presence of deoxynucleotide triphosphate (15m) of The purified segment containing the segment ori of 3 and the restriction enzyme 5roR Cohesive ends of both fragments of plasmid pHC79 cleaved with I and Sal I Smooth the edges. The ligation was carried out using a fragment containing the ori segment of M13 and a fragment containing the ori segment of M13. In equimolar proportions of fragments containing COS segments of medium age λ, 50 mM T ris-)lcj2 (pH 7,5), 10mM MgCj! 2.10mM Dithiothreitol, 3mM adenosine triphosphate, phage T4 DNA Buffer comprising 25 A ligase activity units and 50 qr/ml total DNA Perform in 100I. The ligated DNA was transformed into E. coli JM109 using standard methods. to be introduced. The cells were treated with 25R/rn1 ampicillin, 0.1% IPTC. and 2% L-agar containing 0.1% X-ga1 dye. Ru. Isolate DNA from 10 blue colonies by standard methods ~107-) , and restriction enzymes EcoR1, HindIIl, PstI, BaaI, Analyze using Bgl II and Sal l. Restriction enzyme map as expected (No. 1a) and further manipulated using a construct called diphasmid SKI. do. To reduce the size, 5N SKI was used with restriction enzymes old nd and Bgl. II (5 active units each) and then the anti-ampicillin Qualitative gene-containing fragments were purified by electrophoresis using low melting point agarose. Ru. At the same time, using the same restriction enzymes, the 5pst DN of phage M13mp18 was A and the small molecule fragments are isolated from low melting point agarose after electrophoresis. . Both fragments were mixed with 50mM Tris-HCj2 (pH 7,5), 10mM MgC. 12, 10mM dithiothreitol, 3+nM adenosine triposphate, In 0.1 m72 of a solution containing 5 active units of DNA ligase from phage T4, Ligate at a total concentration of A fragments of 5/mn. Using 10d of this solution, E. coli JM 109 cells were transformed, and the transformed cells were transformed with 25 n/ml of amperage. Picillin, 0.1% (DX-gal and 0.1%<7) containing IPTC Inoculate onto agar. From the blue colony, cells were treated with ampicillin (10k/m7 The cells were grown in 10 mf of medium containing Isolate the DNA (~10 times).

0.5R DNA with restriction enzymes EcoRI, PstI, Hindm, B am I.

Cleaved with Bgl II, Sal I, and electrolyzed in an agarose gel. Analyze by pneumophoresis. It has the expected restriction map (Figure 1a) and SK2 and The named construct is used in the next step.

The DNA of Nismid pHC7905 was cleaved with restriction enzyme Bgl II, The fragment containing the cos segment was then separated from low melting point agarose after electrophoresis. Elute. In addition, the DNA at 2 of SK2 was hydrolyzed with Bglm, and this A preparation of 50mM Tri-s-HCj! (pH 7,5), 10mM MgCf 2.10mM dithiothreitol, 3mM adenosine triphosphate In a buffer containing 5 activity units of DNA ligase of phage T4, the cos segment was isolated. using a 2-fold molar excess of the fragment and bringing the total concentration to ~5/ Ligate using rrLl DNA. Using 10 m of this solution, E. coli JM10 9 cell lines were transformed and then the cells were transformed with ampicillin (25 g/m7'). Inoculate onto Mushiichi agar. from grown colonies on a nitrocellulose filter. A replica was taken and labeled with 32p by nick translation. Hive of Nismid pHC79 with BglIf fragment containing COS segment Perform redization. Are there 5 colonies that hybridize with this COS fragment? cells in 10 ml of medium containing ampicillin (~100 n/me). The DNA (~10 tails) was grown and isolated by alkaline lysis, 0.5x The DNA was digested with restriction enzymes EcoR1, Pat I, Hindm, Bam I, Cleaved with BglII, SalI and electrolyzed in an agarose gel. Analyze by electrophoresis. All five diphasmids have the expected restriction enzyme maps ( 1b), one of which (SK6) will be used in the next step.

Five sword DNA (SK6) was partially hydrolyzed with restriction enzyme EcoRI, and The linear diphasmid is purified by electrophoresis in low melting point agarose.

The resulting preparation was mixed with 20mM Tris-HCj! (pH 7,4), 5+nM MgCf 2.10rnM2-mercaptoethanol, 50mM NacJ, All four types of deoxynucleoside triphosphates, each containing 15z, DNA 1 of polymerase E (2 in 45 n of a solution containing 5 active units of the lenow fragment) Treat for 20 minutes at a temperature of 0°C. Next, add 500mM Tris-H to this solution. (1? (pH 7,5), 100mM MgCj! 2.100mM Dithios Reitol, 30mM adenosine triphosphate and phage T4 DNA recovery. Add Buffer 5I containing 20 gauze activity units. Ligation for 14 hours at 16℃ conduct.

This solution of 10I was used to transform cells of E. coli strain JM109, and then this cell The cells were treated with ampicillin (25x/if), X-gal (0.1%) and IPTG. (0.1%) and inoculated onto agar. Threads from 5 blue colonies Cells were grown in 10 ml of medium containing ampicillin (10/-). Then, isolate the diphasmid DNA (~10R) and treat the DNA with restriction enzymes. EcoRI, Pst I.

)1indII1. Cleaved by Bam I, BglIII, Sal I and analyzed by electrophoresis in an agarose gel. 5 types of analysis Two of the DNAs are the same as the original SK6, and three are tetracycline. does not contain an EcoR1 site in front of the Ron promoter, so these do not contain the expected EcoR1 site. It was proven that the restriction map (Figure 2b) has a restricted map (Figure b).

This diphasmid was named 5K18'. DNA at 0.5 of 5K18' completely hydrolyzed by restriction enzyme Acc, and located outside the polylinker. The cohesive end of the Acc 1 site was clenowed in the presence of dATP and dTTP. Fill with fragments. This preparation was mixed with 50mM Tris-HCj2 (pH 7, 5), 10mM MgCj? 2.10mM dithiothreitol, 3mM adhesion DNA of phage T4 in 50 d of solution containing nosine triphosphate! treated with J gauze (5 activity units) overnight at 16°C and used 10 d of this solution. E. coli JM109 cells were transformed with ampicillin (25ff /mf), X-gal (0.1%) and IPTG (0.1%). Inoculate onto agar. Cells from six blue colonies were treated with ampicillin (100z The DNA was grown in a 10-ml medium containing Isolate by relysis (~10K). 0.5 this DNA with restriction enzyme Acc I and analyzed by electrophoresis in agarose. 4 pieces The lasmids are the same as 5K18' and two contain only one Acc1 site. It has been proven that This diphasmid was named 5K18 (Fig. i2). This was used in the next step.

Diphasmid 5K18 DNA was hydrolyzed with restriction enzyme Bgl II, and The reaction is stopped by heating at 65° C. for 15 minutes. Next, add 3 DNA Dilute twice and self-concatenate.

The ligated DNA was used to transform E. coli JM109 cells, and the cells were then transformed into E. coli JM109 cells. are inoculated onto agar agar containing the antibiotic ampicillin. 10 multiplied cocoons Cells from Ronnie were grown in 10 ml of medium containing ampicillin, The DNA is then isolated by alkaline lysis. 0.5 DNA with restriction enzyme Ec oRI.

Bam I (I, Sal 1. Cleaved with BglII and 1% a Analyze by electrophoresis in a garose gel. The six phasmids are controlled as expected. One of these phasmids is called 5K2A, and the production of λSK9 is used for manufacturing. Phasmid 5K2A is a restriction located outside the polylinker Enzymes EcoRI and Acc 0) Due to the absence of recognition sites, phasumi Do SK2 is different. Therefore, phasmid 5K2A is a segment of replication initiation and an amplicon. a fragment of plasmid pBR322 carrying the gene for stability against syrin, and Pack the polylinker, gene 1acz and single-stranded DNA into the protein of phage M13. A fragment of phage M13mp18 containing a segment for caging. Ru. The size of 5K2A is approximately 4.5X 1000 base pairs (t, n, p,) be.

Next, the recognition site for restriction enzyme Sal I in plasmid 5KZA is removed. centre The DNA of asmid 5K2A was hydrolyzed with the restriction enzyme Sal I, and 65 The reaction is stopped by heating for 15 minutes at a temperature of .degree. DN from E. coli All four deoxynucleotides were synthesized using the Klenow fragment of A polymerase. Blunt the sticky ends in the presence of dotriphosphate. Next, dilute the DNA solution 3x and perform self-concatenation. The ligated DNA was transferred to E. coli JM109 cells. to be introduced. Cells were treated with ampicillin at 25R/ml, 0.1% IPTG and Inoculate Petri dishes with 2% L-agar containing 0.1% X-gal. 10 DNA was isolated from several white colonies according to standard methods and the restriction enzyme Ec oRI.

Analyze using Bam Hl and Sal I. Contains EcoR1, Bam I However, it does not contain the restriction enzyme recognition site SalI and is called SK2ΔS. The phasmid is used in the next step where the restriction enzyme EcoRI of phasmid SK2ΔS is remove the recognition site. For this purpose, restricting the DNA of the phasmid SK2ΔS Hydrolyzed with the enzyme EcoRI and heated for 15 minutes at a temperature of 65°C. The reaction is stopped by Using the Klenow fragment, all four deoxy Blunt the sticky ends in the presence of nucleoside triphosphate. Next, this I Dilute INA 3x and perform self-ligation. Treat the ligated DNA with restriction enzyme 8c. cleaved using oRI and introduced into E. coli strain JM109, and the cells were Inoculate onto agar containing 5R/mpicillin. From 10 colonies, Isolate DNA according to standard methods and use restriction enzymes EcoRI, BamI and analyzed by SalI. Contains Bam I but ECORI and Assembling using a phasmid that does not contain Sal I (this is called SK2ΔSR) A converted phasmid gSR is produced. DNA of phage λgtn with restriction enzyme Bam I and the enzyme is inactivated by heating. Then phage λ The cohesive ends of annealed and removed the central Bam I-enzyme with a size of approximately 6.5 kbp. ．． Purify by gel electrophoresis in 7% low melting point agarose (Preparation A). Fa DNA of Sumid SK2ΔSK was hydrolyzed with restriction enzyme Bam I and heated. The DNA is ligated with Preparation A after inactivating the enzyme. linked dna was introduced into E. coli JM109 cells, and ampicillin (25 gg/mf) was introduced into E. coli JM109 cells. Contain and inoculate onto agar. Proliferated colony Kalanitrocellulose filter Take a replica on top and label with 32p by nick translation Hybridize with Preparation A containing the DNA. Hybridize with said preparation A. Cells from 5 colonies were treated with ampicillin (L containing 10 μl/ml). - grown in 10 d of medium and isolated the plasmid DNA using standard methods. Let go. The DNA thus obtained was analyzed by EcoR1, Bam I, and Sal. Integration of the segment λgt■ into the phasmid SK2ΔSR using the I restriction enzyme Check cloning. This phasmid, called gSR, was used to develop phage vectors. λgeS7 is prepared (Fig. 3). DNA of phasmid gSR is digested with restriction enzyme B. hydrolyze with gl II and inactivate the enzyme by heating. Phage λ EMBL3 DNA was hydrolyzed with the restriction enzyme Baml, and the enzyme was purified. Activate.

Both DNA preparations are mixed in equimolar amounts and ligated. Fold the linked DNA λ protein in vitro, and the resulting phage particles were packaged in vitro. Inoculate a plot of cells of E. coli strain LE392 containing 1% X-gal.

Generate phages from 10 blue plaques, isolate DNA, and restrict Analyze with enzymes EcoRI, Barn II, and Sal I. λgES7 One of the phages, called phage, has the expected structure (Figure 3). This phage Used in the next step.

The DNA of phage λEMBL3 was hydrolyzed with restriction enzyme Bao+1, and The insert fragment, which has a size of approximately 13.7 kbp, was placed in 0.7% low melting point agarose. (Preparation B). DNA of phage λgES7 Hydrolyzed with the restriction enzyme Bam I and the “arms” placed in low melting point agarose. (Preparation C). Mix Preparations B and C in equimolar ratios. combine and connect. The ligated DNA is packaged into the protein of phage λ in vitro. and the resulting phage particles were plotted on cells of E. coli strain LH392. to be inoculated. Phage were generated from six plaques, DNA was isolated, and Analyze with restriction enzymes BcoR1, Baa+1 and SalI. Suggested here The phage vector λSK9 is isolated. This phage vector is shown in Figure 4. It has the mechanism shown.

Example 2. Human genome [lNA fragment cloning in λSK9 3) Cleave the DNA with the restriction enzyme Bam I under standard conditions. The enzyme is inactivated by heating at a temperature of 65° C. for 15 minutes. DN of 3 AλSK9 was hydrolyzed with restriction enzymes Bam old and EcoRI and heated. The enzyme is further inactivated. Mix both preparations together to combine vector DNA and genome. Mass ratio with DNA 1:1), total concentration of IINA ~250R/ml Concatenation is performed as 2. The ligated DNA of lXl0-'g is converted into protein of phage λ. 11,500 of the resulting phage particles were packaged in vitro in E. coli. Inoculate a plot of strain 0359. Fragments from 6 randomly selected plaques L-medium 107 containing phage LE392! let them multiply within, and from them Isolate the DNA. Electrophoretic analysis of this DNA shows that these DNA inserts are all recombinant and have a size of 10-15 kbp. Contains. DNA of one of these (phage N(11) 0.5JQl! is hydrolyzed using 1 unit of restriction enzyme Sal for 30 minutes. Freeze/Touch Freeze 3 The enzyme is inactivated by repeating the procedure several times. Dilute this DNA and bring it to room temperature. Connect for 1 hour. 0.1N of the ligated DNA was transferred to the compete of E. coli JM109. cells and incubated at 0°C for 10 minutes, then at 38°C. After incubating for 5 minutes at a temperature of Incubate for an additional 45 minutes at 50°C. Next, 0.2mj? amperage the cells of Inoculate agar agar containing Syrin (25 μl/ml). Cells from 3 colonies were grown in 10 ml of medium containing ampicillin (50 g/mff). , and I) the NA is isolated by alkaline elution. Electrophoretic analysis of DNA shows The plasmid reportedly has the same DNA insert as the original phage Nα1. Ru.

Industrial applicability The vector λSK9 proposed here can be assembled into a genomic library according to the “classical” method. intended for making lee. Vector λSK9 is the “arm” of phage λ The plasmid After infection of E. coli cells containing the plasmid with helper phage M13, the plasmid The code is packaged into the protein of phage M13 and transferred into a single-chain form. Baek The 5′ and 3 ’-terminus, and on the other hand, the 9-terminus in the IJ noncar The presence of recognition sites for different restriction enzymes facilitates substantial physical mapping of the insert. proceed.

Engraving (1 yen each without change) Engraving (no changes to the content) /:E, 7b Engraving (no changes to the content) Engraving (no changes to the content) /i’-E to l)/? I, , fp-fph I.

I-NiIMkuchi-notzl L butt 3 Engraving (no changes to the content) fE, 5 Procedural amendment (formality) %formula% 1.Display of the incident PCT/SU89100204 2. Name of the invention Address: Shizuko Toranomon Building, 8-10 Toranomon-chome, Minato-ku, Tokyo 105 Phone: 504 -07215, date of correction order December 11, 1990 (shipment date) 6. Subject of correction (1) Translation of the description and claims (2) Drawing translation (FIG, la, lb, 2, 3.5) 7. Contents of amendments (1) Translation of the description and claims (no change in content) (2) Drawing translation (no change in content) 8. List of attached documents (1) Translation of the specification and claims: 1 copy each (2) Translation of drawings: 1 copy international search report

Claims (1)

[Claims] A phage vector λSK9 for producing a genomic library, comprising: 47 ．． 7t. n. p. and; 20.3t. n. p. has a size of Cos-Sal I-DNA fragment of phage λEMBL3-left arm ;3.8t. n. p. Sal I-B of Fasmid SK2A, which has the size of gl II-DNA fragment (EcoR I site is destroyed upon completion of the method and the fragment Asmid SK2A was generated from vector SK18, which contains the DNA of phage λ fragment, produced from phage M13 and cosmid pHC79); 13.7t. n. p. Bam I- of phage λEMBL3 with a size of Bam I-DNA fragment; 0.7t. n. p. Bam I- of the phasmid SK2A having the size of Bgl II-DNA fragment (Sal I site has been removed upon completion of the method) ); 9.2t. n. p. of the phage λEMBL3-light arm, with the size of Sal I-Cos-DNA fragment; recognition of restriction enzymes Bam I and EcoR I The capacity of the vector is 4.4-19.0t. n. p. and the spectrum of the E. coli strain that is the vector host. Kutle is LE392=F', had R514 (rk-, mk-), supE44, su p F58, lac Yl OZ (lca I ZY) 6, gal K2, ga l T22, met Bl, trp R55; J. H. 109=recΔ1,Δ1ac pro, end Al, gyr A9 6, thi-l, hsd R17, sup E44, rel Al, F', tr a D36, pro AB+, lac IoZ, ΔM15; Q359=hsd Rk-, had Mk+, sup F, φ80, P2; All Union Collection of Industrial Microorganisms Sums of the USSR Research Institute for Senate Microorganisms and Industrial Microorganisms Pleading ( All-Union Collection of Industrial M icroorganisms of the USSR Research In institute for Genetics and IndustrialM icroorganism Breeding) and pH No. 19 as 687 Deposited on July 21, 1989; A vector characterized by: