KR100453279B1 - a vector wherein each of 3' end has a projected base same or noncomplementary to each other, EclHKI cassette, and methods for using them - Google Patents

Abstract

The present invention relates to a vector, an Ecl HKI cassette, having a single base overhang at both 3 'ends of the same or non-complementary, and a method of using the vector and the Ecl HKI cassette.

When the cloning vector is prepared using four kinds of Ecl HKI cassettes prepared according to the present invention, unlike the conventional T-vector preparation method, pSL-T is produced by one restriction enzyme reaction and gel extraction (Gel Elution). , pSL-G, pSL-C, and pSL-A vectors can be produced, thereby reducing the loss rate in the manufacturing method. Furthermore, these vectors can bind to PCR products that other conventional T-vector products cannot bind, which can significantly improve cloning efficiency. In addition, the vector prepared by the present invention exhibits excellent cloning efficiency, such as a result of a low number of blue colonies containing no PCR product.

Description

A vector wherein each of the 3 'end has a projected base same or noncomplementary to each other, EclHKI cassette, and methods for using them}

The present invention relates to a vector, an Ecl HKI cassette, having a single base overhang at both 3 'ends of the same or non-complementary base, and a method of using the vector and the Ecl HKI cassette.

Recently, a technique for amplifying a specific gene using a specific primer (hereinafter, referred to as a "PCR amplification primer") for engineering a gene has been remarkably developed. This technique is a technique called polymerase chain reacrion (hereinafter referred to as "PCR") of genes and is used as an essential technique for gene amplification and manipulation. Furthermore, the PCR product is intended to be cloned into a cloning vector for the purpose of reengineering and safely storing DNA molecular sieves (hereinafter referred to as "PCR products") resulting from PCR reactions using specific PCR amplification primers. There were many attempts. The PCR reaction is carried out by a polymerase called Taq. DNA polymerase, which has the property of adding adenine (dAMP) nucleic acid base to both 3 'ends of the PCR result. Therefore, since the PCR result has adenine (dAMP) residues at both ends, the T-vector which binds thymine (dTMP), a base capable of complementary binding to the adenine (dAMP) base, at both 3 'ends is used as a cloning vector. Currently used (see FIGS. 1 and 2).

However, recently, experimental results have shown that the degree of binding of adenine (dAMP) base residues to the 3 'end of the PCR product varies depending on the nucleotide sequence specificity of the PCR amplification primers (Hu, DNA Cell Biol). 12, 763 (1993); Magnuson et al., BioTechniques 21, 700, (1996)). That is, if adenine (dAMP) is present at the 3 'end of the amplified PCR result, the tag DNA polymerase is prevented from binding to one adenine (dAMP) nucleic acid residue, while other nucleic acid residues, that is, guanine (dGMP), The reaction that binds cytosine (dCMP) and thymine (dTMP) occurs more predominantly. In addition, sequences favoring binding of adenine (dAMP) bases (eg, 5'-GTTC ..) according to the base sequence at the 5 'end of a primer for PCR amplification, whereas those which are not preferred (eg For example, 5'-TCAG ..) is present. Furthermore, some of the base sequences that do not prefer adenine (dAMP) remain blunt ends that do not bind any nucleic acid residues, or may be cytosine (dCMP), guanine (dGMP), The bases of thymine (dTMP) are each expressed in the form of the 3 'end. That is, PCR products amplified by the PCR method are bound to adenine (dAMP) base at the 3 'end, or in addition to the base of cytosine (dCMP), guanine (dGMP), thymine (dTMP). Therefore, the existing T-vector having only one thymine (dTMP) nucleic acid residue at the 3 'end cannot be efficiently conjugated to such a variety of PCR outputs, thereby causing an important problem of poor cloning efficiency. In addition, even though there is no problem on the vector as a conventional T-vector, it was confirmed that the PCR results are well cloned, and the PCR results are poor. This phenomenon indicates that the nucleotide sequence of the primer for PCR amplification affects the residue portion of the 3 'end, thereby affecting the cloning of the T-vector.

On the other hand, the T-vector conventionally used is a single thymine (dTMP) nucleic acid residue bound to both 3 'ends of the vector after cleaving the plasmid with a restriction enzyme. That is, to prepare a conventional T-vector, first, the plasmid DNA is subjected to a process of purifying only linear DNA by processing a restriction enzyme that generates a blunt end such as Eco RV, and then the 3 'end of the blunt end. Ethimine (dTMP) residues are bound by terminal deoxynucleotidyl transferase and subjected to another purification process. Therefore, this T-vector production method requires two steps of cleavage and conjugation reactions, so that the loss of the vector of the finished product is not only increased and the recovery is reduced, but also a large amount of thymine (dTMP) base and two enzymes Enzymes and terminal deoxynucleotidyl transferases are consumed, and two purification processes are required, so high manufacturing costs are required. Furthermore, in the conventional T-vector manufacturing method, since the blunt end is made and the thymine (dTMP) residue is attached, self ligation is induced so that the protein translation frame shift even when no PCR result is inserted. There is a problem that white colony appears by causing translation reading frame shift.

Therefore, the present inventors have attempted to develop a high-performance vector system that improves the cloning efficiency for PCR products while solving the above problems.

As a result of repeated studies on vectors which achieve the above object, the inventors have not only thymine (dTMP) nucleic acid residues but also adenine (dAMP), guanine (dGMP) and cytosine (dCTP) nucleic acid residues at the 3 'end of the vector. When the cloning vector is used in a mixed manner, the cloning efficiency of the PCR result was found to be complete, thereby completing the present invention. That is, the present invention, unlike the cloning vectors developed so far, four kinds of nucleic acid residues (nucleotide residues) by binding to both ends of the cloning vector, respectively, and by using the resulting four kinds of cloning vectors, 3 The PCR products, which may have all nucleic acid residues (A, T, G, C) at the end, allow for ligation into cloning vectors. Furthermore, the inventor devised an Ecl HKI cassette in order to easily produce the four types of vectors.

1 is a map of a typical T-vector.

FIG. 1A is a pGEM ^R -T cloning vector and FIG. 1B is a pGEM ^R -T easy cloning vector.

Figure 2 schematically shows the form of ligation of a typical T-vector and PCR result. The two vectors shown in FIG. 2 are one stranded linear double stranded DNA.

3 shows a restriction site of Ecl HKI.

Here, arrow (↓) is a cutting position of Ecl HKI.

4 is a schematic diagram of an Ecl HKI cassette.

Figure 5a shows an Ecl HKI cassette nucleotide sequence and restriction sites are designed to thymine (dTMP) residue occurring at the end of the 3 'region that is cut into the Ecl HKI.

Figure 5B shows fragments after cleaving the Ecl HKI cassette (T) with Ecl HKI restriction enzymes.

Figure 6, Ecl HKI a cassette base sequence designed such that a guanine (dGMP) residue occurring at the end of the 3 'region that is cut into the Ecl HKI.

Figure 7, Ecl HKI is a cassette nucleotide sequence is designed so that the cytosine (dCMP) residue occurring at the end of the 3 'region that is cut into the Ecl HKI.

Figure 8, Ecl HKI is a cassette nucleotide sequence is designed so that the adenine (dAMP) residue occurring at the end of the 3 'region that is cut into the Ecl HKI.

9 is a process for preparing four types of vectors having nucleic acid residues of non-complementary thymine (dTMP), adenine (dAMP), guanine (dGMP), and cytosine (dCTP) at the 3 'end of a straight double-stranded DNA vector. It is shown. Where N is one of the nucleic acid residues of thymine (dTMP), adenine (dAMP), guanine (dGMP), cytosine (dCTP).

10 shows four types of cloning vectors pSL- having nucleic acid residues of non-complementary thymine (dTMP), adenine (dAMP), guanine (dGMP) and cytosine (dCTP) at the 3 'end of a straight double-stranded DNA cloning vector. Schematic representation of T, pSL-G, pSL-C, and pSL-A.

FIG. 10A is a pSL-T vector map, FIG. 10B is a pSL-G vector map, FIG. 10C is a pSL-C vector map, and FIG. 10D is a pSL-A vector map.

The present invention provides a linearized double strand vector having an unpaired single overhang identical or non-complementary at both 3 'ends.

The present invention also provides an Ecl HKI cassette for easily producing the vector.

Furthermore, the present invention provides a method for efficiently inserting a DNA molecular sieve using the vector.

Hereinafter, the present invention will be described in detail.

The present invention, by designing a cassette containing a restriction site (restriction site) of a specific restriction enzyme to bind to the parent vector, by treating the bound vector with the same restriction enzyme, one of the protruding 3 'end of the cut A cassette is prepared to allow the base residue to come out. Restriction sites were selected as Ecl HKI restriction sites to prepare such cassettes. However, the Ecl HKI restriction site can be replaced with another restriction site, as long as one base residue protruding from both 3 'ends cleaved upon restriction enzyme treatment.

The sequence of restriction sites of Ecl HKI is as follows (FIG. 3):

5 'GACNN N ↓ NNGTC 3'

3 'CTGNN ↓ N NNCAG 5'

N in the restriction region is a nucleic acid residue optionally selected from A, C, G, and T. Provided that the N residues are to be paired with complementary base pairs.

On the other hand, when the restriction region of Ecl HKI was treated with Ecl HKI, fragments with the arrow (↓) cut off and one residue ( N in bold and underlined) protruded on the 3 'side were produced. Accordingly, the present invention using the characteristics of the restriction sites of these Ecl HKI, Ecl HKI is to prepare the cassette containing the restriction sites Ecl HKI. Treatment of Ecl HKI devises a sequence of N to yield one desired overhanging adenine (dAMP), cytosine (dCMP), guanine (dGMP), or thymine (dTMP). However, in order for the 3 'ends of both cleaved Ecl HKI restriction sites to be paired with base residues having the same or not complementary to each other, it is preferable to position two restriction sites consecutively in the Ecl HKI cassette. At this time, the second is intended to be a single Ecl HKI restriction site Ecl HKI restriction site on the left of which is N and Ecl HKI restriction site on the right is the N target nucleic acid bases of the base of the upper. On the other hand, the other N in the Ecl HKI restriction site may be preferably selected when creating a unique restriction enzyme site, adjusting the GC ratio, or adjusting the ratio of A and T and G and C.

Below, we will look at the structure of the Ecl HKI cassette in which the 3 'end of each cleaved during Ecl HKI enzyme treatment may have the same protruding one base residue as T, G, C, A, respectively (see Figure 4). .

The Ecl HKI cassette contains two consecutive sequences of the Ecl HKI restriction site. In addition, the restriction region different from the Ecl HKI restriction site is included on both sides of the two Ecl HKI cleavage sites so that the Ecl HKI cassette can be inserted into the existing vector. For example, pGEM vectors, pPUC vector, pBluescript vector existing in order to be able to insert the Ecl HKI cassette in both the cloning vector, the two Ecl restriction sites such as on either side of the HKI restriction site and the Sac II or Pst I, such as It is preferable to include. However, in the Ecl HKI cassette, the Sac II or Pst I restriction sites can be replaced with various restriction sites such as Hind III, Sph I, Apa I, Nde I and the like depending on the intended use.

In addition, the Ecl HKI cassette may add a spacer sequence between two Ecl HKI restriction sites on the cassette to increase the efficiency of the Ecl HKI cleavage reaction. Ecl HKI restriction enzymes have been reported to not function properly when one restriction site is cut and the other restriction site is cut if the same cleavage site is present. Therefore, in order to prevent such a phenomenon, it is preferable to add one or more bases which are next to each other beside the restriction site when devising a primer for forming a cassette. In this case, the spacer may be lengthened by inserting an arbitrary base that does not generate a restriction site present in the parent vector or the cassette.

Furthermore, in order to prepare a vector having a base residue protruding at the 3 'end, a vector containing an Ecl HKI cassette was selected, and the PCR product was inserted when the PCR product was inserted after the vector was treated with an Ecl HKI restriction enzyme. It is necessary to select this inserted vector. Therefore, the Ecl HKI cassette of the present invention is designed to be consistent with the open reading frame of the LacZ gene, which is partially present in the parent cloning vector, so that the LacZ gene can be fully expressed. It is desirable to. This is used to form blue colonies in LBP plate medium treated with IPTG and X-gal when the LacZ enzyme is expressed. For example, vectors inserted with an Ecl HKI cassette can form an intact Rock Jet (Lac Z) gene to express the Rock Jet (Lac Z) enzyme, so that the strain transformed with the vector is IPTG and Blue colonies are formed in X-gal treated LLB plate medium. Furthermore, strains transformed with Ecl HKI restriction enzymes and transformed with the vector inserted with the PCR product form white colonies. Therefore, whether or not the PCR result is inserted can be selected by the color of the colony. That is, if a vector containing no cleavage reaction contains a small amount in the vector into which the PCR result is inserted, it shows blue colony, so that it can be easily identified with the white clone colony represented by the vector into which the PCR result is inserted. have. On the other hand, the linear vector cleaved by Ecl HK restriction enzyme but not inserted into the PCR result does not appear in the medium because it does not transform the strain.

Ecl HKI cassette according to the present invention, in addition to the Rock Jet (LacZ) gene, a variety of reporter genes commonly used for screening, such as GFP (green fluorescence protein) gene, luc (luciferase) gene, lambda lethal gene ( Lambda lethal genes, such as can be inserted into a lethal gene that can destroy the transformed host can be applied as a cloning vector.

It contains two Ecl HKI restriction enzyme sites and a spacer, and generates the same base with both protruding 3 ′ ends during Ecl HKI enzymatic treatment, and a portion of the LacZ gene for the desired vector selection. Ecl HKI cassette comprising Sac II and Pst I restriction enzyme sites for easy insertion into the parent vector, ST-1 and ST-2, ST-3 and ST designed with sequences of SEQ ID NOs: 1-8 -4, ST-5 and ST-6, and ST-7 and ST-8 primers ("cassette forming primers"). However, the sequence of the primer for forming the Ecl HKI cassette is not limited thereto as long as the object of the present invention can be achieved.

Ecl HKI cassettes can be prepared by inducing a hybridization reaction by incubating the primers for forming ST-1 and ST-2 cassettes of SEQ ID NOs: 1 and 2 at 100 ° C. and slowly cooling them.

[SEQ ID NO: 1; ST-1]

5'-GGACCGTGAGTCTTTGACAGAGAGTCTGCA-3 '

[SEQ ID NO: 2; ST-2]

5'-GACTCTCTGTCAAAGACTCACGGTCCGC-3 '

Ecl HKI Cassette (T) (FIG. 5A)

5'-GGACCG T GAGTCTTTGACAGAGAGTCTGCA-3 '(ST-1 primer)

3'-CGCCTGGCACTCAGAAACTGTC T CTCAG-5 '(ST-2 primer)

----- -------- ------- ----

Sac II Ecl HKI (spacer) Ecl HKI Pst I

When the Ecl HKI cassette (T) (FIG. 5a), which is made of primers for forming ST-1 and ST-2 cassettes, is treated with Ecl HKI, two inner Ecl HKI cleavage sites are located at the 3 'end of one thymine residue ( dTMP) is projected (see FIG. 5B).

5'-GGACCG T (3 ') GAGTCTTTGACAGA GAGTCTGCA-3'

3'-CGCCTGGC ACTCAGAAACTGTC (3 ') T CTCAG-5'

Therefore, after inserting the Ecl HKI cassette into the cloning vector, cutting it with Ecl HKI, and removing the middle cut fragment by a gel extraction method, a cloning vector with one residue (T) protruding at the 3 'end is obtained. can do.

When treating Ecl HKI, primers for forming SG-1 and SG-2 cassettes consisting of the sequences of SEQ ID NOs: 3 and 4, which are designed to yield one protruding guanine (dGMP), are identical to the Ecl HKI cassette (T) method. Reaction produces an Ecl HKI cassette (G) (see FIG. 6).

[SEQ ID NO: 3; SG-1]

5'-GGACCGGGAGTCTTTGACAGCGAGTCTGCA-3 '

[SEQ ID NO: 4; SG-2]

5'-GACTCGCTGTCAAAGACTCCCGGTCCGC-3 '

In addition, the treatment for the formation of SC-1 and SC-2 cassettes consisting of the sequences of SEQ ID NOs: 5 and 6 designed to yield one protruding cytosine (dCMP) upon treatment with Ecl HKI is the same as the Ecl HKI cassette (T) method. In response to prepare an Ecl HKI cassette (C) (see FIG. 7).

[SEQ ID NO: 5; SC-1]

5'-GGACCGCGAGTCTTTGACAGGGAGTCTGCA-3 '

[SEQ ID NO: 6; SC-2]

5'-GACTCCCTGTCAAAGACTCGCGGTCCGC-3 '

In addition, the treatment for the formation of SA-1 and SA-2 cassettes consisting of the sequences of SEQ ID NOS: 7 and 8 designed to yield one protruding adenine (dAMP) upon treatment with Ecl HKI is performed in the same manner as the EclHKI cassette (T) method. Reaction produces an Ecl HKI (A) cassette (see FIG. 8).

[SEQ ID NO: 7; SA-1]

5'-GGACCGAGAGTCTTTGACAGTGAGTCTGCA-3 '

[SEQ ID NO: 8; SA-2]

5'-GACTCACTGTCAAAGACTCTCGGTCCGC-3 '

Four types of Ecl HKI cassettes made from the primers for forming cassettes corresponding to SEQ ID NO: 1 to SEQ ID NO: 8 are designed to have restriction enzymes such as Sac II and Pst I because the restriction enzymes Sac II and Pst I sites are designed to exist at both ends. Can be inserted into any cloning vector that has been cut.

Further, parent vectors into which the Ecl HKI cassette is inserted include pGEM vectors (Promega), pPUC vectors (New England Biolabs), and pBluescript vectors (New England Biolabs). The parent vectors are vectors for cloning, including ori genes and antibiotic mark genes (primarily aphicilin, tetratracycline resistant genes), useful cloning sites (e.g., Apa I, HindI II, Genes required for cloning, such as Nco I, Eco RI, Sac I, BamH I, Pst I, Nde I (see FIG. 1: pGEM-T vector map). In addition, the parent vector into which the Ecl HKI cassette is inserted can be used as a DNA library vector and a protein expression vector, depending on the intended use. The protein expression vector is a vector that can be directly expressed by cloning the PCR product, and preferably has a powerful expression promoter such as a tac promoter or a T7 promoter.

Looking at the method of producing a cloning vector using the Ecl HKI cassette as follows. After cutting with Sac II and Pst I insert four kinds of Ecl HK cassette according to the invention cut in the same Sac II and Pst I in the pGEM-5zf (Promega Corporation) vector, respectively, methods such as three Brook (Sambrook et al Plasmid vectors pSL-Te, pSL-Ae, pSL-Ce, and pSL-Ge are prepared by experiment of transformation and colony screening according to Cold Spring Harbor Lab.Press, New York, 1989). The plasmid vectors pSL-Te, pSL-Ae, pSL-Ce, and pSL-Ge according to the present invention were treated with restriction enzymes Ecl HKI ( promega ), and then cleaved, using a gel elution method. To purify the desired cloning vector (FIG. 9). Here, the gel extraction method is the electrophoresis of the Ecl HKI restriction enzyme reactant with 1% agarose gel to extract only the corresponding cloning vector site and the extraction kit (Promega, cat. # A7170). Purification process using the c), the process and the time required to complete the cloning vector is short, so the possibility of contamination of impurities is low, and the purity is high, thus maintaining the high activity of the cloning vector. Can be.

Through the preparation process, 3'-T, 3'-A, 3'-G, and 3'-C non-complementary bases protrude at the 3 'ends of the cloning vector. A, pSL-G and pSL-C cloning vectors can be prepared (FIG. 10).

Cloning vectors such as pSL-T, pSL-A, pSL-G, and pSL-C prepared according to the present invention may be used to clone PCR products protruding one non-complementary nucleic acid residue, but are not limited thereto. The cloning vectors prepared according to the present invention may be applied to transform with a foreign gene, and may also be applied to prepare a DNA library.

Furthermore, Ecl HKI by cassette devise for appropriately selected cassettes to form a N of Ecl HKI restriction site primer when produced, Ecl HKI restriction enzyme the 3 'end of both the cutting during the process to create a single overhanging nucleotide residues of which do not have the same And cloning vectors having these characteristics can be prepared.

[Example 1; Ecl HKI Cassette Production]

₄ μl of 100 pmol primers ST-1 and ST-2 (SEQ ID NOs: 1 and 2), 10 μl of 10 × oligonucleotide buffer (0.1 M NaH ₂ PO ₄ ), and 82 μl of tertiary distilled water, respectively. After the addition was made 100μl and reacted for 10 minutes at 90 ℃. Then, slowly cooled down to room temperature in a styropole box. MgCl ₂ was carefully mixed with 100% ethanol to a final concentration of 10 mM, and then centrifuged at 14000 g for 20 minutes. The resulting reaction was dried and suspended using 10 μl of tertiary distilled water. From this, an Ecl HKI cassette (T) to which primers ST-1 and ST-2 were bound was prepared.

Instead of primers ST-1 and ST-2, primers SG-1 and SG-2 (SEQ ID NOs: 3 and 4), SC-1 and SC-2 (SEQ ID NOs: 5 and 6), SA-1 and SA-2 ( SEQ ID NO: were prepared and are Ecl HKI cassette (T) and the same method as Ecl HKI cassette (G), Ecl HKI cassette (C), Ecl HKI cassette (a) except for using the 7 and 8).

[Example 2; pSL-Te, pSL-Ae, pSL-Ce, pSL-Ge production]

Each of the Ecl HKI cassettes prepared in Example 1 were treated with Sac II and Pst I and purified. In addition, pGEM-5zf (Promega) vector was treated with Sac II and Pst I as a parent vector and purified. 1 μl of the parent vector (50 ng) and 1 μl of 10 × ligand buffer in each 1 μl of the Ecl HKI cassette having “cohesive end” through annealing reaction in Example 1, T4 0.5 μl of DNA ligase and 6.5 μl of tertiary distilled water were treated and reacted at 4 ° C. for 8 hours. In this way, each Ecl HKI cassette and the vector were inserted through a ligation reaction to prepare plasmids pSL-Te, pSL-Ae, pSL-Ce, and pSL-Ge.

[Example 3; Selection of colonies transformed with pSL-Te, pSL-Ae, pSL-Ce, pSL-Ge]

Prepared by the CaCl ₂ method of Sambrook (Sambrook et al., Cold Spring Harbor Lab. Press, New York, 1989, p.1.82-1.84), the prepared JM109 competent strain (competent cell) of Example 2 Transformed with the gated reaction. That is, 10 μl of ligation reactants were mixed with 200 μl of the prepared strain, and then reacted with ice for 30 minutes, and then reacted at 42 ° C. for 90 seconds. Then, after reacting with ice for 1 to 2 minutes, 800 µl of SOC medium was added and incubated for 45 minutes at 37 ° C. 20 μl of these were treated in IPTG and X-Gal treated LB plate media and then incubated at 37 ° C. for at least 12 hours. Plasmid vectors inserted with an Ecl HKI cassette can form an intact Rock Jet (LacZ) gene to express the Rock Jet (LacZ) enzyme, so that the strains transformed with the vector are IPTG and Xgal (X-). Blue colonies are formed in Gal treated LLB plates. Therefore, after incubation in 1ml Elbi (LB) medium containing ampicillin blue colony (Blue colony) with Isu Seeds and incubated for 10 hours at 37 ℃, Promega Wizard plus mini-prep. The plasmid pSL-Ae, pSL-Te, pSL-Ce, and pSL-Ge were extracted using the kit. Some of the extracts were subjected to a cleavage reaction with an Ecl HKI restriction enzyme and analyzed by agarose gel. As a result, it was confirmed that the extract was a vector into which the Ecl HKI cassette was inserted.

[Example 4; pSL-T, pSL-G, pSL-C, pSL-A Cloning Vector Preparation]

Treatment of plasmid vectors pSL-Ae, pSL-Te, pSL-Ce, pSL-Ge, 20 µl, 1 µl Ecl HKI (Promega), 3 µl of reaction buffer E (buffer E), and 6 µl of distilled water at 37 ° C The reaction was carried out for 4 hours. Then, two Ecl HKI cleavage sites present in the Ecl HKI cassette are cleaved by the Ecl HKI enzyme, and the corresponding fragments are cut out between the Ecl HKI cleavage sites. Therefore, the resulting reactions were analyzed with 1% agarose gel, and each DNA band where the enzymatic reaction was completed, i.e., pSL-T, pSL-G, pSL-C, and pSL-A cloning vectors, was cut out with a knife. Then, using Promega's Clean-up Kit, the gel was extracted by dissolving the gel and binding the vector to a special resin. That is, each DNA band was placed in a 1.5 ml test tube, and then treated with 1 ml of resin to react at 37 ° C. for 5 minutes to dissolve the gel. After placing the reactant on a mini column, the solution was removed using a vacuum pump, and the resin with the bound vector remained on the column, followed by washing with 2 ml of 80% isopropanol. Finally, 50 µl of tertiary distilled water was treated in a column, and then transferred to a new experimental tube to obtain a clean cloning vector by spin centrifugation for about 5 seconds. The obtained cloning vectors pSL-T, pSL-G, pSL-C, and pSL-A were quantified using a spectrometer (WPA).

<Test Example 1>

PSL-T, pSL-G, pSL-C, pSL-A cloning vectors prepared using the Ecl HKI cassette according to the present invention and pGEM-T vectors of Promega (cat # A3600; 3 'both ends of base residue T as a control) In the phosphorus vector (see FIG. 1), the efficiency as a cloning vector was compared by the following method.

Using a spectrometer (WPA, Inc.), the concentrations of the vectors were equally 50 ng, respectively, and a 300 bp PCR product was conjugated with the ligation reaction. Then, after transforming according to Sambrook et al. (Sambrook et al., Cold Spring Harbor Lab. Press, New York, 1989), IPV and X-Gal treated ELB ( LB) incubated for 12 hours at 37 ℃. Colony results shown in the Elby (LB) medium are shown in Table 1.

Strains transformed with the vector inserted with the PCR result are shown as white colony. Therefore, plasmids were extracted from the strains inoculated with some of the white colonies, and then digested with Sac II and Pst I. At this time, the agarose gel analysis resulted in a DNA site of about 300bp length was confirmed, from which white clones can be confirmed that the PCR result is inserted.

On the other hand, as shown in Table 1, the pSL-T, pSL-G, pSL-C, pSL-A vector according to the present invention is a blue colony (PCR) without inserting the PCR product than the conventional pGEM-T vector (Blue colony) The number was small. In other words, it can be seen that the method of purifying the vector using the Ecl HKI cassette is much better.

The total number of white colonies (PCR product inserted) was similar to that of pGEM-T vector and pSL-T vector. However, comparing the pSL vectors with each other, one can observe that the PCR output is cloned at a ratio of about 7: 4: 4: 3 (pSL-T; -G; -C; -A). Therefore, when introducing the PCR resultant used in Test Example 1 into the PCR cloning vector, each of four types of cloning vectors at a ratio of pSL-T: pSL-G: pSL-C: pSL-A = 7: 4: 4: 3. When prepared by mixing, high efficiency PCR cloning vectors can be obtained. In other words, in the mixed pSL-T, pSL-G, pSL-C, and pSL-A vector systems, the 3 'ends of the PCR result that the conventional T-vectors could not bind were 3'-C, 3'-G. In addition, PCR results of 3'-A can be combined to maximize cloning efficiency.

Furthermore, specific PCR products may have a different ratio of base residues at the 3 ′ end depending on specific primer sequence characteristics. Therefore, by sampling a portion of a specific PCR result (sampling) to calculate the mixing ratio of pSL-T, pSL-G, pSL-C, and pSL-A cloning vectors in the same manner as above, and then the pSL- mixed with the mixing ratio. Cloning the specific PCR product using T, pSL-G, pSL-C, and pSL-A vector systems can increase cloning efficiency.

Therefore, in view of the above results, it can be seen that the four-base vector using the Ecl HKI cassette shows a much better effect than the conventional vector.

According to the present invention, if a cloning vector is prepared using four new Ecl HKI cassettes, unlike the conventional T-vector production method, pSL-T is performed by one restriction enzyme reaction and gel extraction technique. , pSL-G, pSL-C, and pSL-A cloning vectors can be produced, thereby reducing the loss rate in the manufacturing method. Furthermore, these vectors can bind to PCR products that other conventional T-vector products cannot bind, which can significantly improve cloning efficiency. Moreover, the vector prepared by this invention shows the outstanding cloning efficiency, such as the result which the blue colony which does not contain a PCR result becomes low.

<110> Seoulin Scientific Co., Ltd <120> a vector wherein each of 3 'end has a projected base same or nonc omplementary to each other, EclHKI cassette, and methods for usin g them <130> pa00048 <160> 8 < 170> KOPATIN 1.5 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 1 ggaccgtgag tctttgacag agagtctgca 30 <210> 2 <211> 28 <212 > DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 2 gactctctgt caaagactca cggtccgc 28 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 3 ggaccgggag tctttgacag cgagtctgca 30 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 4 gactcgctgt caaagactcc cggtccgc 28 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 5 ggaccgcgag tctttgacag ggagtctgca 30 <210> 6 <211> 28 <212> DNA < 213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 6 gactccctgt caaagactcg cggtccgc 28 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 7 ggaccgagag tctttgacag tgagtctgca 30 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> a primer for EclHKI cassette <400> 8 gactcactgt caaagactct cggtccgc 28

Claims (17)

In the PCR cloning method using a vector prepared by inserting a DNA molecular sieve, The DNA molecular sieve is a straight double-stranded, both 3 'end has the same or non-complementary base overhang of adenine (dAMP), guanine (dGMP), cytosine (dCMP) and thymine (dTMP) , The vector is a linearized double strand vector having an unpaired single overhang that is the same or non-complementary at both 3 'ends, The base protuberances are made of four types of vectors forming base pairs complementary to the protruding bases of one base protuberance at both 3 'ends of the DNA molecular sieve, and the four types of vectors are mixed at an optimal ratio. PCR cloning method characterized in that it is used. delete delete delete delete Consists of two contiguous Ecl HKI restriction sites and a different kind of restriction site from the Ecl HKI restriction site, but the restriction site of the type different from the Ecl HKI restriction site is located on both sides of the two consecutive Ecl HKI restriction sites. Located The Ecl HKI restriction site As, the left side of the restriction site is Ecl HKI Ecl HKI cassette, it characterized in that the top of the right side of the N and Ecl HKI restriction sites are selected to nucleotide N of the object at the bottom. 7. The method of claim 6, Ecl characterized the Ecl sequence of HKI cassette that is designed to match the protein translation frames (open reading frame) of the report gene (reporter gene) present in the parent vector the Ecl HKI cassette is inserted HKI cassette. 8. The Ecl HKI cassette of claim 7, wherein the reporter gene is a lock jet (LacZ) gene. The cassette according to claim 6, wherein the Ecl HKI cassette is a cassette in which a cassette for forming cassette of SEQ ID NO: 1 and a cassette for forming cassette of SEQ ID NO: 2 are hybridized, a cassette for forming cassette of SEQ ID NO: 3, and a cassette for SEQ ID NO: 4 A cassette hybridized with a primer, a cassette for forming a cassette of SEQ ID NO: 5, and a primer for forming a cassette of SEQ ID NO: 6, and a cassette for forming a cassette of SEQ ID NO: 7 and a primer for forming a cassette of SEQ ID NO: 8 Ecl HKI cassette, characterized in that selected from the group consisting of cassettes. Hybridization with other complementary primers can form a double stranded cassette, characterized in that the primer for forming Ecl HKI cassette, characterized in that selected from the group consisting of SEQ ID NO: 1 to 8. A restriction site different from an Ecl HKI restriction site present on both sides of the Ecl HKI restriction site of the Ecl HKI cassette of any one of claims 6 to 9 is cleaved with the restriction enzyme, and the mother vector is also used as the restriction enzyme. A vector into which an Ecl HKI cassette is inserted, characterized in that it is prepared by inserting the cleaved Ecl HKI cassette into a cleaved parent vector. 12. The method of claim 11 wherein the parental vector is a lock jet (LacZ) gene and the Ecl HK cassette is designed to match the open reading frame of the reporter gene present in the parental vector to be inserted. Vector featuring. delete delete In the method for producing a straight double-stranded DNA vector having a single base overhang that is the same or non-complementary at both 3'-end, Claim 6 to claim 9 Ecl HKI according to any one of items cassette of, Ecl HKI restriction sites exist both on the outside Ecl HKI restriction site and is cut and the matrix vector is also the same limit the different types of restriction sites into the restriction enzyme A first step of cleaving with an enzyme and inserting the cleaved Ecl HKI cassette into the cleaved parent vector by ligation reaction; Cleaving two Ecl HKI restriction sites by an Ecl HKI restriction enzyme; And And optionally a third step of removing fragments between the two Ecl HKI cleavage sites. 16. The method of claim 15, wherein when Ecl HKI cassette inserted into the parent vector full report gene (reporter gene) is a protein translation frame of some presence report gene for the parent vector to be inserted into the sequence of the Ecl HKI cassette to be expressed (open a process of devising in accordance with the reading frame is performed before the first step; Transforming the strain with the vector inserted with the Ecl HKI cassette, A method for producing a vector, characterized in that the process of selecting a colony producing an intact report gene product is performed between the first and second steps. delete