JP2002325573A - Vector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an improved vector capable of synthesizing RNA fraction largely deleting extra sequences in vitro. SOLUTION: This vector has a DNA cloning site within 50 bp of the downstream of a promoter sequence of a RNA polymerase and at least one breakage site of the vector in the downstream of the cloning site and the DNA cloning site has a blunt end type restriction enzyme recognition sequence and the breakage site of the vector has a blunt end type or 5'-protruding end type restriction enzyme recognition sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The invention of this application relates to a vector. More specifically, the invention of this application relates to an improved vector useful for preparing an RNA probe or for TA cloning.

[0002]

2. Description of the Related Art In situ hybridization is based on
It is widely used as a method for identifying a target gene (mRNA) expression site in tissue pieces, cells, embryos, organelles, and the like without extracting intracellular DNA or RNA. This in sit
The most frequently used detection method in the u-hybridization method is a method using an antisense strand RNA probe. An antisense strand RNA probe is prepared by using a DNA fragment inserted into a cloning site of a vector as a template, and using an RNA polymerase promoter (T3, T7, SP6
Etc.) to perform a transcription reaction using RNA polymerase to synthesize in vitro.

[0003] A vector is capable of selecting a transformant using drug resistance as an index, has a plurality of restriction enzyme recognition sequences (multicloning site) at a cloning site, and has a cloning site determined by blue-white determination. In addition to the general conditions as a vector such that it is possible to select clones into which the template DNA fragment has been inserted,
An RNA polymerase promoter sequence is present on at least one side of the multiple cloning site,
A that synthesis is possible, primer sets for determining the base sequence from both sides of the insert template DNA are prepared, and the copy number in the host (such as E. coli) are large,
Those satisfying conditions such as easy preparation of plasmid are used. PBleuscript series (STRATAGENE) and pGEM
Vectors such as the series (Promega) are known.

[0004]

When an RNA probe is prepared using a vector as described above, the end point of transcription is determined by cutting the vector in advance with a restriction enzyme,
Allow A polymerase to spontaneously desorb. However, it has been known that when the cut surface is 3'-overhang, the termination of transcription does not occur well, and the RNA polymerase stagnates or causes transcription of the opposite strand. Therefore, during in vitro transcription of RNA fragments, it is necessary to cut the template DNA fragment with a restriction enzyme that gives a 5′-protruding end or a blunt end at the end of transcription (Nucleic Acids Res. 13: 6).
223-6239, 1985). However, typical general-purpose vectors, such as the pBleuscript series, have 3'- It has many protruding restriction enzyme sites. For this reason, when subcloning a template DNA fragment for synthesizing an RNA fragment, a restriction enzyme site at the center of the multiple cloning site must be used. When in vitro RNA transcription is performed from a template DNA fragment inserted into such a central cloning site, the sequence in the long multi-cloning site is necessarily included in the transcribed RNA fragment. Will be included. In in situ hybridization, for example, p
Found on Bleuscript's multiple cloning site
It is pointed out that the sequence having a high GC content has a high homology with the rRNA sequence, and when detection is performed using the RNA probe prepared by the above-described method, the background becomes high and accurate detection becomes difficult. (Anal. Biochem. 2
55: 95-100, 1998, Biotechniques 14: 458-463, 199
3).

The invention of this application has been made in view of the above-mentioned problems of the prior art, and is an improved technique capable of reducing extra sequences contained in RNA fragments synthesized in vitro. The task is to provide vectors.

[0006]

The present invention relates to a first invention for solving the above-mentioned problems, in which a DNA cloning site is cut within 50 bp downstream of a promoter sequence of RNA polymerase and a vector is cut downstream of the DNA cloning site. A vector having at least one site, the DNA cloning site being a blunt-end type restriction enzyme recognition sequence, and the vector cleavage site being a blunt-end type or 5′-projection type restriction enzyme recognition sequence. provide.

[0007] In a preferred embodiment, the vector of the first invention has two types of opposite promoter sequences, and a cloning site and a vector cleavage site are located between these promoter sequences.

[0008] Further, the present application relates to a second invention, namely, RNA
Provided is a vector having substantially only a DNA cloning site within 30 bp downstream of a promoter sequence of a polymerase, wherein the DNA cloning site is a blunt-end type restriction enzyme recognition sequence.

[0009] In a preferred embodiment, the vector of the second invention has two types of opposing promoter sequences, and a DNA cloning site is located between these promoter sequences.

Hereinafter, embodiments of the present invention will be described, and the vector of the present invention will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The vector of the first invention is obtained by converting an RNA fragment (RNA
Probe), which is a circular double-stranded DNA for synthesizing
NA polymerase promoter sequence (T3, T7, SP6, etc.)
Within 50 bp downstream, preferably within 40 bp, most preferably
It has a cloning site for inserting a template DNA fragment within 35 bp. Further, it has at least one vector cleavage site downstream of the cloning site, preferably immediately after the cloning site. RN
When preparing the A probe, the template DNA is inserted into the cloning site, and then the vector cleavage site is cleaved as a transcription termination point, and the RNA polymerase is allowed to act on the in vitro.
Perform tor RNA synthesis.

In a preferred embodiment of the vector of the first invention, the cloning site and the vector cleavage site are located between two opposite types of promoter sequences. In such a vector, an antisense RNA probe for in situ hybridization is synthesized from a + strand of the template double-stranded DNA by one promoter sequence, and a sense RNA from a − strand (control probe) by the other promoter sequence. Can be synthesized.

In the vector of the first invention, the cloning site is a blunt-end restriction enzyme recognition sequence, and the vector cleavage site is a blunt-end or 5′-overhanging restriction enzyme recognition sequence. Examples of blunt-ended restriction enzyme recognition sequences include AccII, AluI, BalI, Dra
I, EcoRV, FspI, HaeIII, HpaI, NruI, PvuII, RsaI, S
Recognition sequences for restriction enzymes such as caI, SmaI, SphI, SspI, and StuI can be used. The 5'-overhanging restriction enzyme recognition sequence includes AccI, AccIII, AcyI, AflII, A
geI, Alw44I, AseI, AvaI, AvaII, AxyI, BamHI, Bcl
I, BglII, BssHII, BstEII, CbiI, EcoO109I, EcoRI, E
coRII, HindIII, Hinfl, MluI, MspI, NarI, NciI, Nco
I, NdeI, NdeII, NheI, NotI, NspV, SalI, Sau3AI, Sa
Recognition sequences for restriction enzymes such as u96I, ScrFI, SpeI, StyI, TaqI, XbaI, XhoI can be used. Since the DNA cloning site is a blunt-end site, blunt-end ligation of the template DNA prepared by PCR amplification or the like is possible, and since the vector cleavage site is blunt-end or 5′-projection, in vitro Separation of polymerase during RNA synthesis is performed efficiently.

Further, the 3′-end of the blunt end of the cleaved cloning site is obtained by a known method (Nucleic Acids Res. 1).
9: 1154, 1991).
It can also be used as a vector for TA cloning of CR products. That is, the Taq polymerase used in the PCR method has a property that it easily adds an adenyl group (A base) at the end when it reaches the 5'-end of the template DNA during the DNA chain extension reaction. As a result, D amplified in PCR
In the NA fragment, an A base protrudes from both 3 'ends of the target gene sequence (double-stranded DNA), and a complementary thymidyl group (T base)
Does not exist. Therefore, a vector having a T base protruding at the 3 ′ end is prepared in advance, and a PCR amplified fragment is ligated to this vector, so that the product after PCR can be directly and irrespective of the sequence of the amplified gene.
It is possible to clone the fragment. Ligation can be performed efficiently by TA cloning, because DNA fragments having an A base protruding at the 3 ′ end and vectors having a T base protruding are not ligated.

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 1) between the T3 and T7 promoters of pRIBO Easy, which is an example of the vector of the first invention. This vector pRIBO Ea
sy is T3 located on one strand of circular double-stranded DNA
It has a cloning site consisting of a blunt-end restriction enzyme recognition sequence (EcoRV) at a distance of 35 bp from the promoter,
Immediately thereafter, the 5'-terminal restriction enzyme recognition sequence (EcoRI and
BamHI). In addition, 31 from the T7 promoter located on the other strand
The EcoRV site at a distance of bp, and immediately thereafter the 5'-
It has a vector cleavage site consisting of a terminal type restriction enzyme recognition sequence (HindIII and XhoI).

This pRIBO Easy vector has the restriction map pBluescript II SK (+/-) (Stratagene) shown in FIG.
This is a vector created by improving the above, and its configuration other than the multicloning site (MCS) is the same as that of the pBluescript vector. However, the pBluescript vector is 77 bp from the T3 promoter to the EcoRV site that is usually used as a template DNA cloning site for RNA probe synthesis,
In contrast to 55 bp from 7 promoters, this pRIBO Eas
y is as short as 35 bp from the T3 promoter and 31 bp from the T7 promoter to the EcoRV site. Also, pBlues
BamHI site in cript vector Between SmaI site, Sa
Regions with high GC content, such as between the lI site and the KpnI site, that tend to be the background are missing. Furthermore, immediately after the cloning site, each has two types of vector cleavage sites, and it is possible to cut the vector using a restriction enzyme recognition sequence that is not included in the template DNA sequence. Therefore, by amplifying the template DNA fragment of the target RNA probe by PCR amplification or the like, subcloning it into the EcoRV site, and cutting the vector at any of the cleavage sites on both sides thereof, an RN containing no extra sequence is obtained.
An A probe and its control probe can be created.

The four types of vector cleavage sites used in the pRIBO Easy vector are selected so that the reaction of the restriction enzyme used can be performed under the same conditions. Also, the pRIBO Easy Vector contains an XhoI site and
It has 3'-overhanging restriction enzyme sites (KpnI and SacI) outside each of the BamHI sites, but these are pBlues
This is a restriction enzyme site that remains when the multiple cloning site (MCS) is amplified by PCR from cript II and modified into the sequence of FIG. 1A, and is not used as a DNA cloning site or a vector cleavage site. Naturally, these KpnI and SacI sites are not a constituent of the present invention. Further, the vector of the present invention comprises pBluescrip
In addition to the t vector, the vector can be prepared by modifying a pGEM series vector or other general-purpose vectors. Alternatively, an arbitrary plasmid DNA can be prepared by adding the above-described constitution.

The vector of the second invention comprises substantially only a DNA cloning site (blunt-end type restriction enzyme recognition sequence) within 30 bp, preferably within 25 bp, most preferably within 23 bp downstream of the promoter sequence of RNA polymerase. Have. In the case of this vector, when amplifying the template DNA by PCR, a PCR primer to which an appropriate restriction enzyme tag has been added is used, and the PCR product is inserted into a cloning site. Is formed. As a restriction enzyme tag to be added to the PCR primer, a blunt-end type or 5′-projection type restriction enzyme recognition sequence is used. Then, after cutting the restriction enzyme site added to the end of the template DNA, in vitro RNA synthesis is performed by allowing RNA polymerase to act.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 2) between the T3 and T7 promoters of pROBO Easy +, which is an example of the vector of the second invention. This vector pRIB
O Easy + has only a cloning site consisting of a blunt-end restriction enzyme recognition sequence (EcoRV) 23 bp from the T3 promoter located on one strand of the circular double-stranded DNA and 19 p from the T7 promoter on the other strand. are doing. And the template inserted into this cloning site
By cutting the vector at the restriction enzyme site added to the end of the DNA, it is possible to synthesize an ideal RNA probe containing almost no extra sequence.

In this pRIBO Easy + vector also, KpnI and SacI sites are present on both sides of the EcoRV site, but these are the restriction enzyme sites remaining when the sequence was modified by PCR-amplifying MCS from pBluescript II. hand,
Needless to say, this is not a constituent feature of the present invention.

In the case of the pRIBo Easy vector of the second invention, since there is no extra restriction enzyme site, the PCR fragment to which the restriction enzyme tag has been added is cut out with the restriction enzyme and transferred to another expression vector or the like. It is also useful as an intermediate vector for replacement. Of course, like the vector of the first invention, it is also useful as a vector for blunt-end ligation or as a vector for TA cloning.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.

[0023]

EXAMPLES Example 1 Construction of Vector The following synthetic oligonucleotides were prepared. F1: 5'-CCTCGAGAAGCTTGATATCGAATTCGGATCCGAGCT-3 '(SEQ ID NO: 3) R1: 5'-CGGATCCGAATTCGATATCAAGCTTCTCGAGGGTAC-3' (SEQ ID NO: 4) F2: 5'-CGATATCGAGCT-3 '(SEQ ID NO: 5) R2: 5'-CGATATCGGTAC-3 '(SEQ ID NO: 6) F1 and R1 were used to create pRIBO Easy, and F2 and R2 were used to create pRIBO Easy +. The synthesized oligonucleotide is
Dissolve in sterile water to become pmol / μl, F1 and R1, and
F2 and R2 were paired and mixed in equal amounts. 80ml
After being immersed in a water bath for 20 minutes to denature the nucleotide chain, the water bath was turned off to gradually cool to allow annealing (DNA strand association). 1 μl (about 1 pmol) of this DNA association product was added to KpnI and SacI
Vector pBluescript II KS + (ST
RATAGENE) 4 μl (about 0.1 pmol), 5 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and Liga
Ligation was performed by adding 5 μl of tion High (TOYOBO) and incubating at 16 ° C. overnight. The solution after the ligation reaction was transformed into E. coli JM109, and cultured on an LB agar medium containing X-gal, IPTG, and carbenicillin. Pick up white colonies and M13
Perform direct PCR using the primer set, and
A clone giving an amplified fragment shorter than the case where script II KS + itself was used as a template was selected. After culturing the selected E. coli clones in an LB liquid medium, the plasmid was purified, and based on this, the insert was sequenced using M13 primers. Those having the designed sequence were recognized as the target clones. pRIBO Easy and pRIBO Easy +
The E. coli carrying the vector was stored at -80 ° C as a suspension of 25% (v / v) glycerol. Example 2: TA cloning The pRIBO Easy vector was prepared according to the literature (Nucleic Acids Res. 19:
1154, 1991), the vector was cleaved with the restriction enzyme EcoRV, and T-addition of the vector was performed with Taq DNA polymerase.

Next, the rice GS1 cDNA clone RGS28 (Plant Mol. Biol. . 1
3: 611-614, 1989) as a template and PCR was performed in the following cycle.

(94 ° C./1 minute; 52 ° C./1 minute; 72 ° C./1 minute) × 30; 72 ° C./10 minutes; 4 ° C. Cooling The product after PCR was subjected to agarose gel electrophoresis ( After Figure 3)
The amplified DNA fragment is subjected to QIAEX II Gel extraction kit (QIEX
AGEN). The collected material is dissolved in sterile water and approximately 200 ng /
μl. To 1 μl of this solution, 1 μl (10 ng / μl) of T-modified pRIBO Easy vector was added, and further 6 μl of sterilized water and 4 μl were added.
l Ligation high (TOYOBO) was added, and a ligation reaction was performed at 16 ° C for 5 hours. The whole amount of the solution after the reaction was introduced into Escherichia coli JM109, and white colonies grown on an LB agar medium containing IPTG, X-gal and 50 μg / ml carbenicillin were picked up. This was subjected to direct PCR from cells using the M13 primer set (FIG. 4), and a clone having a vector in which the target PCR amplified fragment was retained in the insert was selected. After culturing this E. coli clone and purifying the plasmid, ABI Bigdye terminato
The base sequence of the DNA fragment in the insert was decoded by the r cycle sequencing method, and at the same time, the orientation of the DNA fragment inserted as the insert was confirmed. Those whose decoded base sequence matched the target DNA fragment were stored as target clones. Example 3 Preparation of RNA Probe The E. coli clone obtained in Example 2 (the target DNA fragment was pRIBO E
A plasmid having a plasmid retained as an asy vector insert) was cultured in a liquid medium containing carbenicillin, and then the plasmid was purified. Purified plasmid 10μ
g each, restriction enzyme EcoRI (75U), or HindIII (50U)
After protein digestion, the proteins were removed by phenol extraction, and after ethanol precipitation, they were dissolved in 20 μl of sterilized water. Using the thus obtained linear DNA as a template, a DIG-labeled RNA probe was prepared using a DIG RNA labeling kit (Roche Diagnostics). In addition, at the time of synthesis
RNAplymerase uses T3 RN for EcoRI digested template.
A polymerase was converted to T7 RN
A polymerase was used for each. The RNA probe after synthesis was dissolved in 50 μl of DEPC-treated water after ethanol precipitation, and stored frozen at −20 ° C. until use.

[0026]

As described above in detail, according to the present invention, an RNA fragment from which an extra sequence is largely deleted can be obtained in vitro.
o An improved vector that can be synthesized is provided. By using an RNA probe created by this vector, high-precision gene detection by in situ hybridization or the like becomes possible.

[0027]

[Sequence List] <110> Japan Science and Technology Corporation <120> Vector <130> NP01037-YS <160> <210> 1 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400> 1 AATTAACCCT CACTAAAGGG AACAAAAGCT GGGTACCCTC GAGAAGCTTG ATATCGAATT 60 CGGATCCGAG CTCCAATTCG CCCTATAGTG AGTCGTATTA 100 <210> 2 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400> CGAGCTAGAGCTAGAGCTAGAGCTAGAGCTAGAGCTAGAACGAGCTAGAACGAGCTAGAAGCAGAACGAGCTAGAACGAGCTAGAAGCAGAACGAGCTAGAAGG AATTCGCCCT 60 ATAGTGAGTC GTATTA 76 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400> 3 CCTCGAGAAG CTTGATATCG AATTCGGATC CGAGCT 36 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400> 4 CGGATCCGAA TTCGATATCA AGCTTCTCGA GGGTAC 36 <210> 5 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400 > 5 CGATATCGAG CT 12 <210> 6 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonuc leotide <400> 6 CGATATCGGT AC 12 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400> 7 CGGTGCTGAC AAGTCTTTTG 20 <210> 8 <211> 20 <212 > DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide <400> 8 ACTTCTCCCA GCACAAATGC 20

[Brief description of the drawings]

FIG. 1A shows pRIBO E, which is an example of the vector of the first invention.
B is a nucleotide sequence between the T3 promoter and T7 promoter of asy, and B is pROBO Eas
This is the nucleotide sequence between the T3 promoter and the T7 promoter of y +.

FIG. 2 is a structural diagram of pBluescriptII SK used for creating pRIBO Easy vector and pROBO Easy + vector.

FIG. 3 shows the results of PCR amplification of a sequence (703 bp fragment containing 3′-UTR 113 bp) used as a probe using rice GS1 cDNA as a template. When an agarose gel electrophoresis was performed on 3 μl of the PCR product, an almost single band was obtained (lane 2 arrow). Lane 1 shows the molecular weight marker (EcoT14I
Λ DNA).

FIG. 4 shows the results of M1
This is the result of performing direct PCR using three primer sets and selecting an E. coli clone having an insert of the desired length. In the lane marked with *, the fragment of the desired length has been amplified, indicating that the E. coli carries the desired plasmid.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA20 CA04 CA05 DA06 EA04 FA02

Claims (4)

[Claims] 1. A DNA cloning site within 50 bp downstream of a promoter sequence of RNA polymerase and a vector cleavage site at least one downstream of the DNA cloning site.
A vector, wherein the DNA cloning site is a blunt-end type restriction enzyme recognition sequence and the vector cleavage site is a blunt-end or 5′-projection type restriction enzyme recognition sequence. 2. The vector according to claim 1, wherein the vector has two types of opposing promoter sequences, and a cloning site and a vector cleavage site are located between these promoter sequences. 3. A vector having substantially only a DNA cloning site within 30 bp downstream of a promoter sequence of RNA polymerase, wherein the DNA cloning site is a blunt-end type restriction enzyme recognition sequence. 4. The vector according to claim 3, wherein the vector has two types of opposing promoter sequences, and a DNA cloning site is located between these promoter sequences.