JP2003038177A - Method for preparing library of dna having modified target base sequence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of randomly modifying a base sequence in every region of the sequence and controlling degree of randomness connected with the modified site of the base sequence and the modified base sequence in preparation of a DNA library. SOLUTION: In one of a primer pair, a target region is designed using N (mix of A, T, G and C) and a primer (a first primer) combined with a base sequence complementary to a template DNA at the both side of the target region is designed. In the other side of the primer pair, a primer including a base sequence complementary to an adhesive part of the 5' side of the first primer. Polymerase chain reaction is performed using such designed primer pair and a DNA including the target sequence as a template. If necessary, degradation treatment of the template DNA is performed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing a DNA library having a modified target base sequence, a kit for the method, and a DNA library obtained by the method.

[0002]

2. Description of the Related Art Proteins and RNAs, which are biopolymers, have various functions in life activities as end products of DNA. About 4 human genes by genome analysis
Although it has been suggested that there are tens of thousands of genes, none of them have known functions, and about 244,000 genes were reported as unknown genes. Ultimately, the elucidation of the function of these genes will be the immediate task. Furthermore, elucidation of the basic principles and networks of interactions between genes, and by extension the proteins and RNAs that are their products, could be the most effective approach to understanding life.

By spontaneously folding a protein or RNA after translation or after transcription, it can have catalytic activity for the first time as a three-dimensional molecule and can interact with other proteins. The information is originally input into the primary structure such as the amino acid sequence of protein or the base sequence of RNA, and is stored in the genome in the form of the base sequence of DNA. Therefore, in the process of long biological evolution, the sequences of each gene are continuously selected and conserved under the constraint of functional expression of the final product, protein or RNA. In the case of genes that are conserved in various species from bacteria to humans,
The mutation rate of the functionally insignificant region is high, while high conservation is observed in the functionally indispensable region. The highly conserved region is the center of the catalyst, is an important site for the formation of higher-order structures, and is a region that interacts with other molecules. Proven from biological analysis. That is, it is possible to find functional importance in the conservation of the nucleotide sequence of DNA. Regions that are conserved across species have been functionally selected and are expected to be somewhat important sequences.

In order to identify functionally important sequences,
Generally, a mutant in which the base of DNA is mutated is prepared, and it is examined whether or not the mutant retains the function of DNA before mutation. For example, mutations have been introduced into conserved sequences by the site-directed mutagenesis method, the activity and properties of the obtained mutants have been investigated, and it has been investigated whether the mutation-introduced sequences are functionally important. .

Conventionally, Kunk has been used as a site-directed mutagenesis method.
el et al. (Kunkel, TA, (1985) Proc. Natl. Acad.
Sci. USA, 82, 488) was widely used, but since the operation is complicated and time consuming, a method using PCR (Qu
ick Change ^TM Site-Directed Mutagenesis Kit, Stratag
ene) has come to be used favorably. However, when introducing a mutation at one site, it is not realistic to create a mutant in which all 19 other amino acids are substituted,
Usually, a mutant is designed by intentionally determining an amino acid to be converted to some extent, using hydrophilicity, hydrophobicity, electric charge, etc. as an index. In addition, when mutations are introduced into a plurality of sites at the same time, the variation of the mutant is further increased, and in general, when mutations are introduced into N sites, 20 ^N kinds of sequence variations occur. In this case, it is unrealistic to create all mutants by the site-directed mutagenesis method.

Therefore, a DNA library composed of mutants obtained by randomly modifying the nucleotide sequence of DNA was prepared,
A method of selecting functionally important sequences from this DNA library is used. As a method for randomly altering the base sequence of DNA, there has been used a PCR method (Leun method for reducing the accuracy of DNA synthesis by DNA polymerase).
g, DW et. al., Techinique 1: 11-15) and a PCR method using template DNA mutated with a chemical modifier (JP-A-5-192148).

[0007] However, in these methods, since the introduction of mutation depends on the properties of DNA polymerase and chemical modifiers, not only the introduction of mutation in a specific nucleotide sequence of DNA but also the modification It is also difficult to control the degree of randomness (frequency of four types of bases in the base sequence after mutation). Therefore, the DNA library obtained by these methods has a problem in terms of diversity and is not always suitable for efficient selection of functionally important sequences.

In order to select a functionally important sequence from all possible sequences in principle, it is conceivable to create a library in which a specific region is randomized. Such techniques include the cassette mutation method (Wells,
et al., Gene, 34, 315 (1985); Grundstroem, et al.,
Nucleic Acids Res., 13, 3305 (1985)) is used to insert a random mutation-introduced DNA fragment into a plasmid. However, in the cassette mutation method, a mutation cannot be introduced unless there is an appropriate restriction enzyme in the vicinity of the introduction of the mutation, and because a library is prepared by a DNA ligation reaction, it is necessary to construct a sufficient amount of the library. The problem is that it is difficult.

[0009]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and its object is to randomly modify the nucleotide sequence of every region in the preparation of a DNA library. And a method capable of controlling the modification site of the base sequence and the degree of randomness of the modified base sequence. A further object of the present invention is to enhance the synthesis efficiency and facilitate size expansion in the production of a DNA library.

[0010]

Means for Solving the Problems As a result of intensive studies, the present inventors have solved the above problems by designing a special primer in a polymerase chain reaction using a DNA containing a target base sequence as a template. I succeeded in doing so.
The principle of preparing the DNA library in the present invention is as follows.

First, a primer pair as shown in FIG. 1 is prepared (FIG. 1 shows only the case where the first primer binds to the sense strand). One of the primer pairs is a primer (first primer) in which a target region is designed with N (mixture of A, T, G, and C), and a nucleotide sequence complementary to the template DNA is bound on both sides thereof. The other is a primer containing a complementary nucleotide sequence near the 5'end of the first primer.
Design (second primer). Using the primer pair designed in this way, polymerase chain reaction is performed using the DNA containing the target sequence as a template. The target sequence will be a random sequence derived from the first primer. Further, by varying the mixing ratio of A, T, G, and C in the target region of the first primer, the degree of randomness of the target region can be controlled. This makes it possible to target a specific sequence and control the degree of randomness thereof to prepare various DNA libraries according to the purpose.

Based on the above principle, the present inventors have actually prepared a 46-size library in which the ^six bases are randomized with the operon rrnB of Escherichia coli ribosomal RNA gene as a target sequence. succeeded in. We used the template DN
We also succeeded in reducing the number of template DNAs having the target sequence in the library by performing methylation of A, cleavage of methylated DNA, and λ exonuclease treatment.

Next, in order to verify the usefulness of the prepared library in the identification of a functionally important nucleotide sequence, the inventors of the present invention developed the SSER method using the uniquely developed SSER method described later. Library from rrn B
An attempt was made to select a DNA that retains the function of. As a result, rrn B
In addition to the two nucleotide sequences that retain the function of, the sequences actually present in mitochondrial ribosomal RNA and ribosomal RNA of other species were identified.

That is, the present inventors succeeded in producing a DNA library in which a specific nucleotide sequence was randomly modified, and at the same time, identified the nucleotide sequence in which the produced DNA library was functionally important. It was proved to be useful for the present invention, and the present invention was completed thereby.

More specifically, the present invention provides the following (1) to (10). (1) A method for producing a DNA library in which a target base sequence is modified, which comprises performing a polymerase chain reaction using a DNA containing the target base sequence as a template using the primers (a) and (b) below. Including the method. (A) a primer that hybridizes to a region containing a target base sequence, wherein the base sequence in the primer corresponding to the target base sequence is modified from the target base sequence (b) the first primer Second primer containing a nucleotide sequence complementary to the 5'side region of the nucleotide sequence corresponding to the target nucleotide sequence in the primer (2) Methylation of template DNA to be subjected to polymerase chain reaction, (1) . (3) The method according to (2), wherein the template DNA is methylated by treatment with a sequence-specific DNA methylating enzyme. (4) The method according to any one of (1) to (3), which comprises decomposing the template DNA after the polymerase chain reaction. (5) The method according to (4), wherein the template DNA is decomposed by a DNA-degrading enzyme treatment. (6) The method according to (5), wherein the DNA degrading enzyme treatment is a restriction enzyme Dpn I treatment. (7) The method according to (5), wherein the DNA degrading enzyme treatment is a restriction enzyme Dpn I treatment and a subsequent λ exonuclease treatment. (8) A DNA library having a modified target base sequence, which is produced by the method according to any one of (1) to (7). (9) A kit for carrying out the method according to any one of (1) to (7), which comprises at least one of the following (a) to (c). (A) DNA methylase (b) Enzyme that cleaves methylated DNA (c) λ exonuclease

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a “DNA containing a target nucleotide sequence”, which is a template DNA for polymerase chain reaction, is
Any DNA whose nucleotide sequence is desired to be modified can be used. The form of the template DNA may be single-stranded or double-stranded. Further, it may be linear or cyclic.

The "target base sequence" means a base sequence which is a target of modification of bases in a template DNA for polymerase chain reaction. When the DNA containing the target base sequence is a double-stranded DNA, the target base sequence is meant to include both the target base sequence in the sense strand and its complementary sequence in the complementary strand. "Complementary strand" means A: T (but U for RNA), G:
It refers to one strand of a double-stranded nucleic acid consisting of base pairs of C to the other strand.

In the present invention, a polymerase chain reaction is carried out using a primer having a specially designed primer pair with a DNA containing a target base sequence as a template. One of the primer pairs is a primer that hybridizes to a region containing the target base sequence, and the base sequence in the primer corresponding to the target base sequence is a primer (first primer) modified from the target base sequence. is there. The “region containing the target base sequence” means the target base sequence in the template DNA and its 5 ′.
It means the flanking base sequences on the 3'side and the 3'side. In the first primer, the modification site may be a contiguous base in the template DNA, or may be a plurality of bases located apart from each other. Further, the modified base may be one,
There may be a plurality. Considering the size of the DNA library and the number of cells that can be transformed at one time, the number of modified bases is usually 30 bases or less, and preferably 10 bases or less. To increase the number of modified bases while maintaining the size of the library, use A, T, G,
The ratio of C should be changed. For example, if the original base is 70% and the remaining 3 bases are 10% each, the N base library size will be 1 / 0.7 ^N = 1.43 ^N , and the library size when 39 bases are modified. Is
1.43 ³⁹ = 1143156. This is the size of a library that completely randomizes 10 bases (4 ¹⁰ = 1048576 ways).
Is almost equivalent to

For the first primer, if necessary, when the amplification product of the polymerase chain reaction is the original sequence (target sequence), this sequence is derived from the template DNA or selected from random sequences. A mutation may be introduced to distinguish whether it is a substance. When using a DNA library for the identification of functionally important sequences, this mutation is preferably a mutation that does not affect the function of DNA.

The first primer is a mix of A, T, G, C in the target region (if necessary, the ratio of these bases may be varied, or a mix of 2 or 3 of these bases may be used). And the base sequences complementary to the template DNA are arranged on both sides. "Complementary nucleotide sequence" is
Although it is preferably a completely complementary nucleotide sequence, it may not be a completely complementary nucleotide sequence as long as it does not violate the intended purpose of the DNA library to be produced and does not inhibit the polymerase chain reaction. The ratio of complementary base pairs (T to A, C to G) to all the base pairs in the region where the template DNA and the first primer hybridize is usually at least 70%, preferably 80% or more. , More preferably 90% or more, further preferably 95% or more. The oligonucleotide designed in this manner can be synthesized, for example, by the amidite method. The synthesized oligonucleotide can be purified by cutting out from the gel depending on the degree of purification.

When prepared by DNA synthesis, the chain length of the first primer is usually 15 to 150 bases, preferably 20 to 100 bases, more preferably 50 to 6 bases.
It has 0 bases. Also, in the case of randomizing two distant regions, a polymerase chain reaction is performed using a pair of primers with random mutations introduced into each region, and the amplified double-stranded DNA is used as the first and second primers. As a result, a polymerase chain reaction can be performed to prepare a library. The first and second primers in this case are usually 30 to 500 base pairs of double-stranded DNA.

The other one of the primer pairs is a primer (second primer) containing a base sequence complementary to the 5'side region of the base sequence corresponding to the target base sequence in the first primer. The second primer is designed not to include the same base sequence as the target base sequence but to include the base sequence 3 ′ downstream thereof. By doing so, the target base sequence is replaced with the random sequence of the first primer.

The second primer is "the 5'side region of the base sequence corresponding to the target base sequence in the first primer".
It is not necessary to include a base sequence complementary to the entire sequence. A nucleotide sequence complementary to a part of the region may be included as long as it does not inhibit the polymerase chain reaction. The “complementary base sequence” of the first primer in the second primer is preferably a completely complementary base sequence, but it does not violate the intended purpose of the DNA library to be prepared and does not inhibit the polymerase chain reaction. As long as it is not a completely complementary nucleotide sequence. Complementary base pairs for all base pairs of the corresponding region of the second primer with respect to the first primer (A, T, G
The ratio of C) is usually at least 70%, preferably 80% or more, more preferably 90% or more, and further preferably
95% or more. The second primer designed in this way is
For example, it can be synthesized by the amidite method. The synthesized oligonucleotide can be purified by cutting out from the gel depending on the degree of purification.

The chain length of the second primer varies depending on the design of the first primer, but when prepared by DNA synthesis, it is usually 10 to 100 bases, preferably 15 to 50 bases, and It is preferably 20 to 30 bases.

The first and second primers are designed to specifically hybridize with a desired region of the template DNA. The term "specifically hybridizes" means under ordinary hybridization conditions, preferably under stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Labor).
atory Press, New York, USA, 2nd edition 1989 conditions)
Means that it hybridizes with a desired region and does not hybridize with other regions. The first and second primers may be designed so that the first primer hybridizes to the sense strand and the second primer hybridizes to its complementary strand with respect to the template DNA, and vice versa. And the second primer may hybridize with the sense strand.

In the present invention, the polymerase chain reaction using the template DNA and the first and second primers can be carried out, for example, under the following conditions.

10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-H
Cl (pH 8.8), 2 mM MgSO ₄ , 0.2 mM dNTPs, 0.1% Triton X-
100, 1 mg / ml nuclease-free bovine serum albumin, template DNA 10 ng, each primer 125 ng, dNTP mix 5
μl, Pfu Turbo (Stratagene) 1 μl (2.5 U), Pre-heat at 95 ° C for 30 seconds, denaturation at 95 ° C for 30 seconds (Step
1), annealing at 55 ℃ for 1 minute (Step2), extension at 68 ℃
2 minutes / kbp (Step3), then store at 4 ℃. Step 1 to Step 3 are performed for 18 cycles. Also, Step3
The reaction time is calculated from the length of the template DNA using 2 minutes / kbp as a guide. The temperature in Step 2 is calculated from the primer sequence and base composition. More preferable PCR reaction conditions are as follows: template DNA amount is 150 ng and primer amount is 1
Set to 2.5 pg.

When it is desired to reduce the DNA having the original sequence in the amplification product by the polymerase chain reaction, the template DNA is decomposed after the polymerase chain reaction. In order to decompose the template DNA, for example, the template DNA to be subjected to polymerase chain reaction is methylated, and a restriction system (restricti
onsystem) to decompose methylated DNA. Sequence-specific DNA to methylate template DNA
Methylase is used. The sequence-specific DNA methylating enzyme is not particularly limited as long as it can utilize the above-mentioned restriction system in terms of its type and number of uses, but is preferably m-Alu I, m-Hae III, m- It is Hap II. In the present invention, these may be used alone or in combination.

M-Alu I, m-Hae III and m-Hap II are AGCT and GG, respectively. It is an enzyme that recognizes each of 4 bases of CC and CCGG and specifically methylates the 5-position of C like AG ^m5 CT, GG ^m5 CC and ^m5 CCGG, respectively. DNA methylated at these sites is eliminated by the restriction system of Escherichia coli against foreign DNA. Specifically, the gene product of mcrA recognizes and cleaves ^m5 CCGG,
Since B recognizes and cleaves ^Gm5C, all sites methylated by the three enzymes are targeted and eliminated by the restriction system.

On the other hand, it is also possible to decompose the template DNA by performing a DNA-degrading enzyme treatment after the polymerase chain reaction. Usually, DNA adenine methylase (dam) is present in E. coli cells. This enzyme recognizes the GATC sequence and methylates the 6th position of the 2nd A (6-methyladenosine; ^m6A ). Therefore, template D prepared from E. coli
It is known that all NAs are methylated at this position. As an enzyme that decomposes such template DNA, for example, the restriction enzyme DpnI can be used.

In the present invention, the above methylated
In addition to the DNA decomposition treatment, a nuclease treatment such as λ exonuclease can be performed. By carrying out such a treatment, it is possible to decompose a DNA fragment into mononucleotides. It is considered that the DNA fragment generated by DpnI digestion partially forms a complementary strand with the template DNA and is replicated in the cell. In particular, since λ exonuclease uses only DNA in which the 5'end of the DNA is phosphorylated as a substrate, synthetic D
Do not target library DNA amplified from NA. Therefore, the DN methylated by λ exonuclease
Only the DNA fragment generated by the decomposition treatment of A can be digested.

According to the method of the present invention, a DNA library in which a desired base sequence on DNA is randomly modified can be prepared, and furthermore, the degree of randomness of the modified base sequence can be controlled. it can. Further, by decomposing the template DNA, the proportion of DNA containing the modified base in the DNA library can be increased. Therefore, the DNA library prepared by the method of the present invention is
It can be suitably used for selection of mutants retaining the function of the target base sequence and identification of a functionally important base sequence in DNA. The present invention includes a DNA library produced by the method of the present invention and having a modified target base sequence.

The above-mentioned DNA methylating enzyme, enzyme for cleaving methylated DNA, and λ exonuclease can be preferably used for degrading template DNA in the method of the present invention. The present invention includes a kit for carrying out the method of the present invention containing these enzymes alone or in combination. In the preparation of these enzymes, in addition to the enzymes, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protein stabilizers (BSA, gelatin, etc.), preservatives, etc. May be mixed if necessary.

[0034]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] Operon rrn of Escherichia coli ribosomal RNA gene
Preparation of DNA library in which B is randomly modified First, the operon rrnB of E. coli ribosomal RNA gene is
It was incorporated into pMW218 (having a kanamycin resistance gene) having an origin of replication derived from pSC101 and named pRB-Kam ^r (FIG. 2). Then, for the reason described below, pRB-Kam ^r was methylated with a DNA methylase to prepare methylated pRB-Kam ^r . Next, using methylated pRB-Kam ^r as a template, a library was constructed by the PCR method using the first primer and the second primer shown in FIG. The reaction solution shown in Table 1 was prepared and PCR reaction was performed. Specifically, Pre-heat at 95 ° C for 30 seconds, denaturation at 95 ° C for 30 seconds (Step 1), annealing at 55 ° C for 1 minute (St
ep2), extension was performed at 68 ° C. for 20 minutes (Step 3), and then at 4 ° C. for storage. Step 1 to Step 3 were performed in 18 cycles. After completion of the reaction, directly add 10 units of Dpn I and 5 to the reaction solution.
The unit λ exonuclease was added and the template plasmid was digested at 37 ° C for 90 minutes. QIA quick (QIAGEN) clean u
What was done was made into the library. Finally 4.8 ml PCR
Finally, 58 μg of a random library was obtained.

[0035]

[Table 1]

[Example 2] Functional analysis of rrnB using the above DNA library The present inventors performed functional analysis of rrnB using the DNA library prepared in Example 1. Specifically, the present inventors have carried out a functional analysis of rrnB by a method (SSER method) of selecting a functionally important sequence from a DNA library using "selection pressure" of plasmid DNA. went. What is selective pressure?
Multiple plasmid DNAs having the same replicon (origin of replication) cannot coexist stably in the host cell,
Properties of mutual exclusion by selective pressure (Datta, N. 197
9. Plasmid classification: Incompatibility groupin
g. In plasmids of medical, environmental and comme
rcial importance (ed.KN Timmis and A. Puhler),
p3. Elsevier, Amsterdam).

We have created another plasmid, pRB-SacB-Amp ^r . This plasmid is called pSC10
PMW118 with replication origin derived from 1 [Nippon gene company] To rrn
B and Bacillus subtilis Sac B are incorporated. In addition, pRB-Sa
cB-Amp ^r has a drug resistance gene different from pRB-Kam ^r .

The E. coli strain TA542 strain, which was constructed by the group of Squires et al. And which destroyed all seven ribosomal RNA gene operons (rrn) existing in the E. coli genome, was rr
To have a plasmid containing the n operon (pHK-rrnC +) and a plasmid (pTRNA66) into which 5 tRNA genes that have disappeared due to gene disruption of the rrn operon have been cloned, and to complement the function of the gene lost from the genome Is a strain that survives in Japan (Asai et. Al., PNA
S, 96: 1971-1976, 1999). The present inventors have added p
RB-SacB-Amp ^r was transformed, and by selecting with ampicillin, pHK-rrnC ⁺ was eliminated, and a strain (NT101 strain) in which pRB-SacB-Amp ^r was replaced was prepared.

Next, in order to decompose the template DNA, pRB-Kam ^r, which serves as a template for the DNA library, was methylated. Specifically, 50 mM Tris-HCl (pH 8.0), 20 mM NaCl, 10 mM EDT
A, 1 mM DTT, 80 μM S-adenosyl methionine (SAM), pRB
-Kam ^r 50 μg, m-Alu I 50units, m-Hae III 50units, m
-Hap II 50 units were mixed and adjusted to 500 μl with dH ₂ O. 37 ° C
After 30 minutes of reaction, 66 μl of 3M NaCl was added, and the mixture was further reacted at 37 ° C. for 30 minutes. Cleaned up methylated DNA with QIAqui from QIAGEN was used as a PCR template. pRB-Ka
In the case of m ^r , there are 44, 32, and 44 methylation sites for m-Alu I, m-Hae III, and m-Hap II, respectively.
120 points will be methylated. It has been confirmed that the efficiency of the PCR reaction when creating the library is not affected by this treatment at all. In E. coli, all of these sites became cleavage sites for mcrA or mcrCB, and this treatment made it possible to treat the template DNA almost perfectly. Table 2 compares the transformation efficiency of the NT101 strain before and after methylation. It can be seen that the methylation significantly reduces the transformation efficiency of the NT101 strain. In addition, XL1 BlueMR lacking mcrA and mcrCB in the control
Using (Stratagene), it was clarified that the methylation was eliminated by the restriction system because the efficiency was not different by the methylation. It is expected that the amount of template DNA will be further reduced by digesting the template with Dpn I and λ exonuclease after PCR.

[0040]

[Table 2] * XL1 BlueMR (Stratagene); (mcrA) 183, (mcrCB-hsds
MR-hrr) 173

Then, it was prepared by the method described in Example 1.
The DNA library was introduced into the above-mentioned NT101 strain. Specifically, 1.16 μg of the library was added to 400 μl of NT101 strain competent cells prepared by the calcium method, and the cells were placed on ice for 1 hour. Incubate at 42 ℃ for 45 seconds (heat shock), 600 μl
SOC medium was added, and the cells were shake-cultured at 37 ° C for 1 hour (curin
g). LB (spc / kan) agar medium (40 μg / m in normal LB agar medium)
l Spectinomycin and 50 μg / ml kanamycin added. ) And cultured at 37 ° C. for 1 to 2 days. Finally 20.
58 ml of the library was transformed into 8 ml of competent cells, and the cells were plated on 16 square Petri dish agar plates.

Next, the colonies grown on the LB (spc / kan) agar medium were suspended in 200 μl of LB medium, and 5 μl of the LB (spc / kan / SUC) agar medium containing sucrose (LB (spc / kan) (Medium added with 5% sucrose) spotted. In the process, Bacillus subtilis
The cells with Sac B were killed. After culturing at 37 ° C for 1 day, the resulting colonies were able to replace pRB-SacB-Amp ^r with the library.

This colony was inoculated into 1.25 ml of 2 × LB medium and shake-cultured at 37 ° C. for 24 hours. A region extracted from the plasmid and randomized with ABI prism3100
The nucleotide sequence of (2448-2454) was determined. For Sequencing, Forward: 5'CCTTGAAATACCACCCTTTAATG 3 '(SEQ ID NO: 3) and Reverse: 5'TGCTTTCAGCACTTATCTCTTCCG
The 3 '(SEQ ID NO: 4) primer was used.

The original sequence of the randomized region is
ATAACAG (2448-2454), and the second (2449) T can be replaced with C (O'Connor et. Al., Nucl. Acid.
Res, 29: 710-715, 2001), so this position was previously substituted with C on the first primer and used as a marker (specific mutation) after selection. Thus, a library of this region NCNNNNN (N is any base), and 4 ^six (409
6 kinds of sequence variations are obtained. The nucleotide sequence of 63 colonies finally grown on the sucrose plate was determined, and as a result, pRB-Kam ^r was replaced with 38 nucleotides as the background.
25 were selected from the library (ie, two specific mutations derived from the primer were introduced).

The selected ones had any of the following 5 types of sequences. It was found that A2448 can be substituted with all four bases, and A2453 can have a T only when the 2448 position is A. The consensus sequence will be NCAAC (A / T) G. X-ray crystallographic analysis pointed out that it is a catalytic base A
The 2451 was completely preserved. ACAACAG is a sequence common to prokaryote including E. coli, archaeal ribosomal RNA, and most organelle ribosomal RNA. ACAACTG (A245
3T) is the sequence of eukaryotic ribosomal RNA, TCAACAG (A
2448T) is a protozoan mitochondrial ribosome
This is the sequence found in RNA. In this way, the result that the function selection of ribosomal RNA in E. coli can select even the sequences of ribosomal RNA of other organism species such as eukaryote and mitochondria was obtained.

This means that the function of ribosomal RNA is universal across species. Since sequences (A2448G and A2448C) that do not exist in the actual organism were also obtained, this method means that sequences that can function but have been evolutionarily selected are selected. This is a very important finding in analyzing the function of. In addition, when Escherichia coli having these five kinds of sequences were co-cultured, only E. coli having wild-type ACAACAG survived after 48 hours. Therefore, it can be said that the high degree of conservation of this region is maintained by the evolutionary selection pressure. The above results indicate that the method of the present invention can be used for functional analysis and functional modification of DNA. It also shows that the reagents and the like used in the examples can be used as a kit for preparing a DNA library having a modified target base sequence.

[0047]

[Table 3]

[0048]

INDUSTRIAL APPLICABILITY According to the present invention, in the preparation of a DNA library having a modified nucleotide sequence, the site where a mutation is introduced and the degree of randomness of the introduced mutation can be controlled. In addition, the template DNA can be excluded from the DNA library if necessary. Therefore, according to the present invention, various DNA libraries can be prepared under strict control. This makes it possible to prepare a DNA library that meets the purpose, depending on the purpose of use of the prepared DNA library. Further, by applying the DNA library prepared by the method of the present invention to the SSER method developed by the present inventors, the mutation that retains the function of identifying the functionally important sequence in DNA and the function of the target base sequence. Sequences can be efficiently selected.

[0049]

[Sequence list]                                SEQUENCE LISTING <110> tsutomu, suzuki       kimitsuna watanabe <120> Method for DNA library construction with randomized sequence. <130> SEN-A0113 <140> <141> <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <220> <221> misc_feature <222> (20) .. (26) <223> n represents a, t, c, or g <400> 1 atgcatctga ggccccgtan nnnnnntcgg atcgacgatc ataagctta 49 <210> 2 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <400> 2 tacgtagact ccggggcata taacagagcc tagctgctag tattcgaat 49 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <400> 3 tagctgctag tattcgaat 19 <210> 4 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <220> <221> misc_feature <222> (18) <223> n represents a, t, c, or g <220> <221> misc_feature <222> (20) .. (24) <223> n represents a, t, c, or g <400> 4 taaaaggtac tccggggncn nnnngcttat accgcccaag agttcatatc gacggcgg 58 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <400> 5 cccggagtac cttttatccg ttgagcgatg g 31 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <400> 6 ccttgaaata ccacccttta atg 23 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized pri mer sequence <400> 7 tgctttcagc acttatctct tccg 24

[Brief description of drawings]

FIG. 1 N means any base.

FIG. 2 is a diagram showing the expression vector for ribosomal RNA (pRB-SacB-Amp ^r ) used in this example, and the vector for constructing the library (pRB-Kam ^r ) used in this example.

FIG. 3 Underlines indicate specific mutation sites.
N means any base.

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Claims (9)

[Claims] 1. A method for preparing a DNA library in which a target nucleotide sequence is modified, which comprises the following (a) and (b):
A method comprising performing a polymerase chain reaction using the DNA containing the target nucleotide sequence as a template, using the primer of 1. (A) a primer that hybridizes to a region containing a target base sequence, wherein the base sequence in the primer corresponding to the target base sequence is modified from the target base sequence (b) the first primer A second primer containing a base sequence complementary to the 5'side region of the base sequence corresponding to the target base sequence in the primer 2. A template DNA used for polymerase chain reaction.
The method of claim 1, wherein the is methylated. 3. A DNA in which methylation of template DNA is sequence-specific
The method according to claim 2, wherein the method is performed by treatment with a methylating enzyme. 4. The method according to claim 1, which comprises decomposing the template DNA after the polymerase chain reaction. 5. The method according to claim 4, wherein the decomposition of the template DNA is carried out by a DNA degrading enzyme treatment. 6. The method according to claim 5, wherein the DNA degrading enzyme treatment is a restriction enzyme DpnI treatment. 7. The method according to claim 5, wherein the DNA degrading enzyme treatment is a restriction enzyme Dpn I treatment followed by a λ exonuclease treatment. 8. A DNA library having a modified target base sequence, which is produced by the method according to any one of claims 1 to 7. 9. A kit for carrying out the method according to claim 1, which comprises at least one of the following (a) to (c). (A) DNA methylase (b) Enzyme that cleaves methylated DNA (c) λ exonuclease