JP2003159064A - Method for recloning dna fragment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method by which simple and sure recloning can be carried out by forming restriction enzyme cleavage sites capable of ligating to a secondary vector at both ends of an insert DNA cloning into the secondary vector without cleavage with a restriction enzyme. <P>SOLUTION: This method is to reclone the insert DNA cloned into a primary vector to the secondary vector. The method comprises (1) carrying out PCR amplification of a region containing the insert DNA of the primary vector and preparing a double-stranded DNA fragment having the following conditions (a) and (b): (a) at least one end thereof containing a base sequence constituting a cohesive end and (b) absence of a base species n<SB>x</SB>just after a complementary sequence of a base sequence constituting a protruding end of the cohesive end in the complementary sequence, (2) making a T4 DNA polymerase act on the double-stranded DNA fragment in the presence of an excessive amount of the base species n<SB>x</SB>, removing the base sequence on the 3' side from the base species n<SB>x</SB>and providing ends of the double-stranded DNA fragment as cohesive ends and (3) cleaving the secondary vector so that the respective ends of the double- stranded DNA fragment can be ligated and ligating the secondary vector to the double-stranded DNA fragment. <P>COPYRIGHT: (C)2003,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] TECHNICAL FIELD [0001] The invention of this application relates to a DNA fragment.
And a method for recloning. Further details
Alternatively, the invention of this application is cloned into a primary vector.
DNA fragments such as cDNA, etc.
About how to re-clone the vector.
You. [0002] 2. Description of the Related Art As a method of searching for a novel gene, there are two methods.
CDNA synthesized from mRNA transcribed from each gene
A new cDNA is isolated from the set of cDNA libraries (cDNA library),
The structure and function of the protein encoded by the cDNA
An analysis method has been performed. Individuals in the library
Each cDNA is cloned into a vector (primary vector).
Expression of protein from the cDNA, etc.
To do this, the insert cDNA was transferred to another vector (2
To the next vector) (re-cloning)
There is. Conventionally, this recloning method is mainly used.
The following methods (i) and (ii) have been adopted. (i) Derived from the primary vector at both ends of the insert DNA
DNA containing insert DNA using restriction enzyme recognition sequence
Cut out the fragment with restriction enzymes and cut out the secondary vector with the same enzymes.
The DNA fragment is ligated to the cut site. (ii) contains a restriction enzyme recognition sequence present in the secondary vector
PCR amplification of the insert DNA using primers
Cut the product and secondary vector with the same enzyme and insert
The DNA is ligated into a secondary vector. [0004] However, the conventional method
In the case of method (i), the same restriction is conveniently applied to the secondary vector.
Enzyme recognition sequences are not always present. Also, the second vector
If the vector is a fusion protein expression vector, the fusion
-Coding sequence of protein to be toner and insert DN
Whether or not the reading frame matches with A depends on chance
No. In the case of the conventional method (ii),
Sequence of both ends of DNA can be set conveniently.
Therefore, the problem of the conventional method (i) is solved. But
The restriction enzyme recognition sequence at both ends in the sequence of the insert DNA.
If it is included in the product,
The insert DNA itself is also cut. [0006] The invention of this application addresses the problems of the prior art described above.
This was done in light of the points
Cut with restriction enzymes at both ends of insert DNA to be cleaned
System that can be ligated to a secondary vector without
Easy and reliable recloning by forming a restriction enzyme cleavage site
The challenge is to provide new ways to enable
ing. [0007] SUMMARY OF THE INVENTION This application is based on the above-mentioned object.
As an invention to solve the above, the primary vector
Reclosed insert DNA into the secondary vector
(1) Importing the primary vector
PCR-amplify the region containing the insert DNA and perform the following conditions (a)
And (b): (a) a salt in which at least one end constitutes a sticky end
Including a base sequence; (b) Complementary sequence of base sequence constituting sticky end protruding sequence
In the column, the base type n immediately after_xDoes not exist, with 2
Prepare a double-stranded DNA fragment and (2)
And excess base species n_xT4 DNA polymerase in the presence of
Act on the base species n_x2 bases after removing the 3 'base sequence
(3) Secondary vector
Each end of the double-stranded DNA fragment
Cleavage is made possible, and double-stranded DN is added to this secondary vector.
DNA fragment characterized by ligating A fragment
Provides a method for recloning DNA. Hereinafter, embodiments will be described, and the method of the present invention will be described.
This will be described in detail. [0009] DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention comprises the following steps:
Inserter DNA into a double-stranded DNA with specific conditions
Step of PCR amplification as a fragment; (2) a small amount of double-stranded DNA fragment
Making at least one sticky end; and (3) at least
A double-stranded DNA fragment with a sticky end at one end
Cloning process. The following
The process will be described.Process (1) In this step (1), a region containing the insert DNA of the primary vector is included.
Primers that anneal to the region before and after the
Oligonucleotide)), using the primary vector as a template
To perform PCR (polymerase chain reaction)
And the following conditions (a) and (b): (a) a salt in which at least one end constitutes a sticky end
Including a base sequence; (b) Complementary sequence of base sequence constituting sticky end protruding sequence
In the column, the base type n immediately after_xDoes not exist, with 2
This is a step of preparing a single-stranded DNA fragment (PCR product). The double-stranded DNA fragment obtained in this step (1)
For example, it has a base sequence as shown in Sequence Example 1. Sequence example 1: 5'-w₁w_Twow_Threew_Fourw_FiveNNNNN ------ NNz₁-3 ' 3'-x₁x_Twox_Threex_Fourx_FiveNNNNN ------ NNy₁-Five' In Sequence Example 1, "NNNN --- NN" is the PCR-amplified
Insert DNA sequence, "w₁w_Twow_Threew_Fourw_Five"Included in PCR primers
Array, "x₁x_Twox_Threex_Fourx_Five"Is its complementary sequence.
The left end of the row is within the frame to satisfy the condition (a).
Indicated "w₁w_Twow_Threew_Fourw_Five"Array and" x_Five"Is the salt that constitutes the sticky end
It has a base sequence. And "w₁w_Twow_Threew_Four"At the sticky end
"X" complementary to this projection sequence₁x_Twox
_Threex_Four"X for array"_Five"The same base species does not exist (Article
(B)). Also, "y" on the 3 'side of the second strand (lower stage)₁"Primer
Is the base contained in₁"Is its complementary base.
Therefore, the right end of this sequence constitutes a blunt end. Such a base sequence is shown in Sequence Example 2 below.
The PCR method using the PCR primer set as shown
Amplification and preparation. Sequence example 2:     Forward primer: 5'-w₁w_Twow_Threew_Fourw_Fivennn--nn-3 '     Reverse primer: 3'-nn--nnny₁-Five' In this primer sequence, "nnn--nn"
Base sequences corresponding to both ends of the insert DNA sequence
You. Such primers have a synthetic primer of about 15 to 50 bases.
It can be designed as a oligonucleotide. Constituting the adhesive end at the left end in Sequence Example 1
The base sequence to be used is, for example, a restriction enzyme as shown in Table 1.
This is an array in the frame of the knowledge array. Other than the sequences shown in Table 1
Can use various restriction enzyme recognition sequences
You. In addition, the protruding arrangement of the sticky ends was as shown in Sequence Example 1.
Does not need to be a four-base sequence,
Length that can be ligated to other sticky ends (2 base sequence
Above). Furthermore, as described later, this viscosity
The terminating end is a sequence existing in the existing restriction enzyme recognition sequence.
Need not be. [0013] [Table 1] In Sequence Example 1, the blunt-ended base
Vs. "y₁-z₁"Can be any combination. However,
In order to perform step (2) efficiently,_Five"And" z₁"Is the same
It is preferably a base species. If "x_Five"And" z₁Is different
If the base type is _Five"And" z₁"But all" x₁x_Twox
_Threex_Four"It is necessary not to be present. The following sequence example 3 shows that after cutting EcoRI at the left end,
Has the base sequence (in the frame) that constitutes the sticky end of
Has a blunt-end CG base pair (SmaI cleavage site)
It is an example of a double-stranded DNA fragment. Sequence example 3: 5'-AATTCNNN ----- NNG-3 ' 3'-TTAAGNNN ----- NNC-5 ' In the method of the present invention, the amount of double-stranded DNA
The reason that both ends are sticky ends is that both ends are blunt ends
In step (3), insert into the secondary vector
This is because the transfer efficiency becomes extremely poor.
This is because there is a risk of the insertion direction being reversed. On the other hand, in arrangement example 4, adhesive ends are formed at both ends.
It is an example of a double-stranded DNA fragment having a base sequence to be formed. Sequence example 4: 5'-w₁w_Twow_Threew_Fourw_FiveNNNNN ------ NNz₁z_Twoz_Threez_Fourz_Five-3 ' 3'-x₁x_Twox_Threex_Fourx_FiveNNNNN ------ NNy₁y_Twoy_Threey_Foury_Five-Five' The base sequence (sequence in the frame) that constitutes the sticky ends of both ends is
Various existing restriction enzyme recognitions, including the various sequences shown in Table 1.
It can be appropriately selected from the knowledge sequence. However, both ends
Base sequences that constitute the cohesive ends of each part are different
It is preferred that In the case of the same sticky end,
To select the correct insertion direction for the secondary vector
Is required. In addition, the process described below
To perform (2), use "x_Five"And z₁Is the same base species
Is preferred. The arrangement example 5 has a different adhesion at both ends.
In the example of a double-stranded DNA fragment having a base sequence constituting the terminal
Yes, bases that constitute the sticky end after AccIII cleavage at the left end
The sequence and the salt on the right end that constitutes the cohesive end after TaqI cleavage
Each has a base sequence. In this case, "x_Five"
And z₁The base species corresponding to "are the same (T). Sequence example 5: 5'-GGCCANNN ----- NNTCG-3 ' 3'-CCGGTNNN ----- NNAGC-5 ' On the other hand, "x_Five"And z₁"When is a different base species,"
x_Five"And z₁"But all" x₁x _Twox_Threex_Four"And" z_Twoz_Threez_Fourz_FiveExists in
It is necessary not to. Sequence Example 6 shows such a double strand
This is an example of a DNA fragment.
The base sequence that constitutes the end is adhered to the right end after AgeI cleavage
It has a base sequence that constitutes a terminal. Sequence example 6: 5'-GGCCANNN ----- NNACCGG-3 ' 3'-CCGGTNNN ----- NNTGGCC-5 'Process (2) In this step, the double-stranded DNA fragment prepared in the above step (1) is used.
(Eg, Sequence Example 2)_Five"Existence
In the presence of T4 DNA polymerase, 3 '→
Using 5 ′ exonuclease activity, base species x_FiveMore 3 '
Base sequence "3'-x₁x_Twox_Threex_Four-5 '"is removed to cut double-stranded DNA
The end of the piece is the sticky end. For example, in the case of the double-stranded DNA fragment of Sequence Example 3
In the presence of excess deoxy GTP (dG),
By reacting remelase, Sequence Example 7 was shown.
Thus, the left end is the sticky end. Sequence example 7: 5'-AATTCNNN ----- NNG-3 ' 3'-GNNN ----- NNC-5 ' In this case, the G-3 ′ at the right end also has an excessive amount of dG,
Eliminates digestion by T4 DNA polymerase and maintains blunt ends
I do. When the right end is C-3 'as in Array Example 8,
Is T4 DNA polymerase in the presence of excess dG and dC
To form cohesive ends and blunt ends
can do. Sequence Example 8: 5'-AATTCNNN ----- NNC-3 ' 3'-GNNN ----- NNG-5 ' Similarly, in the case of the double-stranded DNA fragment shown in Sequence Example 5,
Activate T4 DNA polymerase in the presence of excess dT
As a result, as shown in Sequence Example 9, both ends are different.
A double-stranded DNA fragment having sticky ends is obtained. Sequence example 9: 5'-GGCCANNN ----- NNT -3 ' 3'- TNNN ----- NNAGC-5 ' In the case of Sequence Example 6, the presence of excessive amounts of dT and dA
By acting T4 DNA polymerase in the presence,
The double-stranded DNA fragment shown in Sequence Example 10 is obtained. Sequence example 10: 5'-GGCCANNN ----- NNA -3 ' 3'-TNNN ----- NNTGGCC-5 'Process (3) In this step, the polymerase treatment in the step (2) is performed.
Each end of the double-stranded DNA fragment prepared by
Restriction enzyme for secondary vector to enable ligation
And a double-stranded DNA fragment was ligated into this secondary vector.
To For example, a secondary vector is constructed using EcoRI and SmaI.
By cutting, the double-stranded DNA fragment shown in Sequence Example 7 was cut.
The pieces can be ligated. Similarly, a secondary vector
Is cut with AccIII and TaqI to obtain two of Sequence Example 9.
Secondary vector in which the strand DNA fragment was cut with AccIII and AgeI
Can be ligated to the double-stranded DNA fragment of Sequence Example 10
It becomes. By the above steps (1) to (3), restriction enzyme cleavage is performed.
Insert the end of the insert DNA into a secondary vector
Can be re-cloned into
You. The example shown above is a double-stranded DNA fragment.
Corresponding to the cuts at both ends of the
The case of digestion with a restriction enzyme is shown. However, this
The method described above is based on the cloning site
One has an end that can be ligated.
The basic principle is to prepare a double-stranded DNA fragment.
Therefore, in step (1), a double-stranded DNA fragment to be PCR-amplified
Does not necessarily constitute sticky ends with existing restriction enzymes
It is not necessary to have a nucleotide sequence at the end. For example, FIG. 1 shows that EcoRI and HindIII
Ligation of the double-stranded DNA fragment into the cut secondary vector
This is an example of the operation. The restriction enzyme HindIII is shown in Sequence Example 1.
X in the base sequence_FiveIs x_Four(A), so HindIII
Double-stranded DNA fragment with cohesive end sequence at the end
Can be prepared by the polymerase treatment in step (2).
Can not. Therefore, as shown in FIG.
EcoRI and HindIII
To obtain sequence (B), and then digest with HindIII using Klenow.
One dA at the 3 'end of the end and two dA at the 3' end of the EcoEI stump
In addition, the cloning site is designated as sequence (C). Meanwhile, primary
The vector insert cDNA was prepared using the PCR plasmid shown in sequence (D).
PCR amplified using Immerset and double-stranded DNA of sequence (E)
Prepare fragments. This distribution in the presence of excess dT
Apply T4 DNA polymerase to row (E) and stick at both ends
The terminal sequence (F) is prepared. This array (F) is an array
Ligation can be performed at the cloning site of (C). In the method of the present invention, the secondary vector
End sequence of double-stranded DNA fragment ligated to
PCR primer sequence) is the insert of the primary vector
Design appropriately considering the sequence before and after the DNA sequence
Expression vector (especially fusion vector) as a secondary vector.
When using a synthetic protein expression vector)
Ligation of the strand DNA fragment with the secondary vector
A stop codon is formed or, like proline
Amino acid cod that causes distortion of protein three-dimensional structure
It is necessary to design so that no pattern is formed. The present invention will now be described with reference to examples.
Will be described in more detail and concretely.
The description is not limited by the following examples. [0025] DETAILED DESCRIPTION OF THE INVENTION
Modulin cDNA is used in E. coli, mammalian cells, and yeast.
EcoRI-SmaI cleavage of the mother expression vector (secondary vector)
The site was recloned. 1: Processing of secondary vector The following were used as secondary vectors. E. coli expression vector pET30a (Novagen)
Made) Mammalian cell expression vector pMIK-Neo Yeast two-hybrid system DNA binding domain vector pGBKT7 (Clontech) 1.1: Processing of pET30a and pMIK-Neo EcoRI part of the multi-cloning site of these vectors
There is a blunt-ended restriction enzyme site downstream of the
NotI site is blunt-ended using Klenow fragment
It was end. First, at 37 ° C. with a reaction solution having the composition shown in Table 2,
Vector by treating for 2 hours and at 65 ° C for 20 minutes
Was cut with EcoRI and NotI. [0027] [Table 2] Next, 1 μl each of 100 mM dGTP, 100 mM dCTP
And Klenow (Boehringer) at 37 ° C for 30 minutes
After the reaction and extraction of phenol / chloroform,
Apply to an S-400 spun column equilibrated with ultrapure water and add 1 μl
And stored at −20 ° C. As a result, about 100 times
Of the vector solution was prepared. 1.2: Processing of pGBKT7 This vector has a SmaI blunt-end site downstream of the EcoRI site.
Since it was present, it was cleaved with these restriction enzymes. Destination
First, it was treated at 37 ° C for 2 hours with a reaction solution having the composition shown in Table 3.
By this, the vector was cut with SmaI. [0029] [Table 3] Next, the reaction solution was equilibrated with ultrapure water to form S-
After applying to a 400 spun column, add 11 μl of 10X buffer H and
Add 2 μl of EcoRI and react at 37 ° C for 2 hours.
Is cut with EcoRI and phenol / chloroform is extracted
Then, apply the aqueous phase to an S-400 spun column and dispense 1 μl at a time.
And stored at -20 ° C. As a result, about 110 vectors
A star solution was prepared. 2: Creation of insert cDNA Avian skeletal muscle tropomodulin cDNA (1st strand DNA)
Was used as a template to obtain a double-stranded DNA fragment.
The primer sets are as follows. [0031] Forward: 5'-AATTCATGTCTTACAGAAAGGAGCTAGAGA-3 '
(SEQ ID NO: 1) Reverse: 5'-CTGGGAGCTACACTCCAGTTCTGCACTTGG-3 '
(SEQ ID NO: 2) In addition, these primers phosphorylated the 5 'end. The composition of the PCR reaction solution was as shown in Table 4.
It is. [0033] [Table 4] The PCR reaction was performed by denaturing (98 ° C., 15 seconds) -anneal.
Ring (65 ° C, 2 seconds)-30 cycles of extension (74 ° C, 30 seconds)
I went. This PCR reaction solution was
After Worm extraction, apply the aqueous phase to S-400 spuncolumn
Treated with T4 DNA polymerase in the presence of dGTP (37 ° C,
in 30 minutes). Table 5 shows the composition of the reaction solution. [0036] [Table 5] After the reaction, the phenol / chloroform
After extraction of the rum, the aqueous phase is applied to an S-400 spun column,
And precipitate the precipitate with 10 μl of ddH_TwoDissolved in O
An insert solution was used. By this processing, the arrangement example shown in FIG.
As shown, the left end has a cohesive end after EcoRI cutting, and the right end
A double-stranded DNA fragment having a SmaI site at the end was created. 3: Ligation The vector treated in step 1 and the double-stranded strand prepared in step 2
The DNA fragment was ligated. Composition of the reaction solution
Is as shown in Table 6. [0039] [Table 6] The reaction was performed at 16 ° C. for 30 minutes, and the three types of vectors were used.
A double-stranded DNA fragment was recloned in each case. Each of these vectors contains Escherichia coli DH5
Transform α by a standard method and plate
Cultured. About 2 from the start of PCR to the start of plate culture
Completed in time, compared to the conventional method (about 1 week), the operation time
And the process was greatly shortened. [0042] As described in detail above, the present application
According to the present invention, insert DNA such as cDNA clone
Re-cloning into another vector quickly and conveniently
Becomes possible. This allows for the expression of genes
Efficiency of functional analysis of protein is greatly improved. [0043] [Sequence list] <110> <120> Sub-cloning method for insert DNA <130> NP01287-YS <140> <141> <160> 2 <210> 1 <221> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence: Synthesized Oligonucleotide <400> 1 AATTCATGTC TTACAGAAAG GAGCTAGAGA 30 <210> 2 <221> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence: Synthesized Oligonucleotide <400> 2 CTGGGAGCTA CACTCCAGTT CTGCACTTGG 30

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process chart showing an example of recloning according to the method of the present invention.

Claims (1)

Claims 1. A method for recloning an insert DNA cloned in a primary vector into a secondary vector, comprising the steps of (1) PCR-amplifying a region containing the insert DNA of the primary vector. And the following conditions (a) and (b): (a) at least one end contains a base sequence constituting a sticky end; (b) a complementary sequence of a base sequence constituting a sticky end protruding sequence; It is provided with, base type n _x is not present immediately after the 2
The stranded DNA fragments were prepared, (2) the double-stranded DNA fragment by the action of T4 DNA polymerase in the presence of an excess of base type n _x with respect to, 3 from the nucleotide species n _x 'side of the nucleotide sequence To remove the ends of the double-stranded DNA fragment into cohesive ends. (3) Cut the secondary vector so that each end of the double-stranded DNA fragment can be ligated. Double-stranded DN
A method for recloning a DNA fragment, comprising ligating the A fragment.