JPH11178582A - Amplification of nucleic acid molecule and synthesis of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and efficiently amplifying a single kind of nucleic acid molecule, a method for producing a protein using a synthetic system for a cell-free protein capable of simply synthesizing a sufficient amount of the protein and a method for simply constructing a protein library. SOLUTION: A nucleic acid molecule group containing a nucleic acid molecule to be amplified is isolated and purified to subsequently provide a state in which an amplifiable nucleic acid molecule other than a primer in a reactional solution of a polymerase chain reaction(PCR) is composed of only the objective nucleic acid molecule and the concentration of the primer used is regulated to >=10 nM for one kind of the primer to carry out the PCR in a method for amplifying the objective nucleic acid molecule according to the PCR. Furthermore, a protein is produced by using a template DNA containing a promoter DNA sequence, a ribosome-binding DNA sequence, a DNA sequence capable of encoding the objective protein and a terminator DNA sequence or a variant terminator DNA sequence and having the sum total of the four kinds of the DNA sequences of >=50% based on the total DNA sequences in a method for producing the protein in a synthetic system for the cell-free protein containing a cell-free extract solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for amplifying a nucleic acid molecule, and more particularly to a method for amplifying a single kind of nucleic acid molecule simply and efficiently. The present invention also relates to a method for producing a protein using a cell-free protein synthesis system. The present invention further relates to a method for constructing a protein library using the method for amplifying a nucleic acid molecule and the method for synthesizing a protein.

[0002]

2. Description of the Related Art As a method for amplifying only a single kind of nucleic acid molecule from a group of various kinds of nucleic acid molecules, a method of combining a plasmid with a host microorganism such as Escherichia coli or yeast has been adopted. This utilizes the property that only one molecule of a plasmid is incorporated into a host microorganism such as Escherichia coli or yeast, and the plasmid is introduced into the host microorganism by a calcium chloride method, an electroporation method, etc. Is a method of obtaining a population of microorganisms containing a single type of nucleic acid molecule by isolating and growing the same, and consequently amplifying the single type of nucleic acid molecule. But,
The operation of binding the target nucleic acid molecule to the plasmid, the operation of introducing the plasmid into the host microorganism, the operation of isolating and growing the microorganism, and the like are very complicated, and require advanced technology and a great deal of time and expense. . Particularly, in the field of changing the nucleotide sequence of gene DNA encoding an amino acid sequence, creating a mutant protein having a changed amino acid sequence using the DNA mutant, and creating a more industrially effective protein, It is necessary to isolate different types of DNA molecules into a single type of molecule, which is extremely difficult. In addition, the demand for the cell-free protein synthesis system has been increasing in recent years because the operation up to protein synthesis is very simple as compared with a system using living cells such as living organisms and cultured cells. On the other hand, PCR (polymerase chain reaction) is a very effective and simple DNA gene amplification method.
There is a need for the development of a technology that allows easy and large-scale synthesis of the protein encoded by DNA directly from DNA obtained by PCR without using DNA. A system for performing cell-free protein synthesis using DNA of a PCR product is already known (Proc. Natl. Acad. Sci. USA, Vol. 94, 412-417,
1997). However, conventionally, it has been difficult to synthesize a sufficient amount of protein, and increasing the amount of synthesized protein has been an important issue.

[0003]

The first object of the present invention is to
An object of the present invention is to provide a method for amplifying a single kind of nucleic acid molecule simply and efficiently. A second object of the present invention is to provide a method for producing a protein using a cell-free protein synthesis system, which can easily synthesize a sufficient amount of protein. A third object of the present invention is to provide a method for easily and efficiently constructing a protein library.

[0004]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies, isolated and purified a nucleic acid molecule group containing a nucleic acid molecule to be amplified, and obtained a solution having a molar concentration of the target nucleic acid molecule. By conducting PCR (polymerase chain reaction) in a very low state and using a primer having a sufficiently low concentration, it has been found that only a single nucleic acid molecule of interest can be efficiently amplified, and the present invention has been completed.

According to the present invention, in a method for amplifying a target nucleic acid molecule by PCR, after isolating and purifying a nucleic acid molecule group containing the nucleic acid molecule to be amplified, an amplifiable nucleic acid molecule other than a primer in a PCR reaction solution is obtained. A PC consisting of only the target nucleic acid molecule and the concentration of the primer to be used being 100 nM or less per one kind of primer,
The present invention provides a method for amplifying a nucleic acid molecule, comprising performing R. Here, a single kind of nucleic acid molecule means both DNA and RNA specified by a base sequence. The present invention also provides a method for producing a protein in a cell-free protein synthesis system containing a cell-free extract, wherein the method comprises using a single type of nucleic acid obtained by the method of the present invention as a template. To provide. Here, the cell-free protein synthesis system refers to mRNA
And a cell-free translation system for synthesizing proteins and polypeptides by reading the above information, and a system containing both a cell-free transcription system and a cell-free translation system for synthesizing RNA using DNA as a template. The present invention further provides a method for producing the protein of the present invention, wherein the above-described nucleic acid amplification method of the present invention is separately performed on at least two kinds of nucleic acid molecules, and each of the amplified at least two kinds of nucleic acids is used as a template. It is intended to provide a method for carrying out the method for each nucleic acid and constructing a protein library comprising at least two kinds of proteins respectively encoded by the obtained at least two kinds of nucleic acids.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, nucleic acid molecules to be amplified are DNA and RNA. Hereinafter, each step of the isolation / purification of the nucleic acid molecule group and the PCR in the present invention will be described in detail. i. Isolation and Purification of Nucleic Acid Molecule Group Containing Target Nucleic Acid Molecule The nucleic acid molecule group containing the target nucleic acid molecule used in the present invention includes DNA linked by ligation reaction, DNA synthesized from RNA by reverse transcription reaction, and the like. Any nucleic acid molecule group that can be a substrate of the nucleic acid molecule may be used. The isolation / purification of the nucleic acid molecule group varies depending on the material used, but any ordinary method may be used. Specifically, any method generally used such as HPLC (high-performance liquid chromatography), electrophoresis, a biotinized primer / avidin method, a primer fixing tube method, and the like may be used. However, it is necessary to use a method capable of effectively isolating and purifying the target nucleic acid molecule without damaging the target nucleic acid molecule. Unless the target nucleic acid molecule group is isolated and purified, amplification of a nucleic acid molecule having no similarity to the target nucleic acid molecule often occurs. Therefore, in the present invention, it is necessary to remove nucleic acid molecules other than the target nucleic acid molecule that may be contained in the sample by isolating and purifying the target nucleic acid molecule group. Examples of nucleic acid molecules other than the target nucleic acid molecule contained in the sample include, for example, laboratory equipment,
When a sample is prepared by amplifying contaminated DNA derived from a hand or the like and a nucleic acid molecule of interest by PCR, primers and the like used at that time are exemplified. In the present invention, a nucleic acid molecule group consisting of nucleic acids having substantially the same molecular weight can be obtained by isolating and purifying a nucleic acid molecule group containing a nucleic acid molecule to be amplified.

Ii. Method of Using Only Nucleic Acid Molecules of Interest In the present invention, various methods can be used as means for making the amplifiable nucleic acid molecules other than the primers in the PCR reaction solution to consist only of the nucleic acid molecules of interest. The simplest method is to isolate and purify a nucleic acid molecule group containing and then dilute it. Easily by dilution, the number of amplifiable nucleic acid molecules other than primers in the PCR reaction solution is changed to one or two or more molecules of a single type of molecule, that is, the amplifiable nucleic acid molecules other than primers are converted to the target nucleic acid. The state can be made up of only molecules. Upon dilution, it is desirable to add tRNA or the like in order to prevent nucleic acid molecules from adsorbing on the tube wall. The appropriate amount of addition is about 0.1 to 10 μg / ml.

Iii. Concentration of Primers in PCR Reaction Solution In the present invention, it is necessary to carry out PCR by setting the concentration of primers in the PCR reaction solution to 100 nM or less for one type of primer, preferably 10 nM.
It is desirable to perform PCR as follows. When the concentration of the primer exceeds 100 nM, non-specific amplification reaction easily occurs between the primers, amplification of the primers such as a so-called primer dimer occurs, the amplification efficiency of the target nucleic acid molecule decreases, and the yield decreases. descend.

Iv. Thermostable polymerase Any one commonly used may be used. A hot start method is desirable for the initiation of the reaction to prevent the formation of primer dimers.

In the present invention, confirmation that a single type of nucleic acid molecule has been amplified is performed by determining the sequence of a nucleic acid obtained by PCR, for example, DNA itself, or a clone of the same into a plasmid. Can be.

Next, a method for producing a protein using a cell-free protein synthesis system containing a cell-free extract will be described. In the present invention, the nucleic acid used as a template is not particularly limited as long as it is amplified by the above-described amplification method of the present invention. However, preferably, the promoter DNA
A template DNA comprising a sequence, a ribosome binding DNA sequence, a DNA sequence encoding a target protein, a terminator DNA sequence or a mutant terminator DNA sequence, and wherein the total of the four DNA sequences is 50% or more of the total DNA sequence. .

A promoter DNA sequence is a DNA sequence to which RNA polymerase binds first during mRNA synthesis, and a ribosome-binding DNA sequence is a DNA at a site to which ribosomes (complexes controlling protein synthesis) bind during protein synthesis. Array, terminator DN
The A sequence or the mutant terminator DNA sequence is a sequence that stops the synthesis of mRNA by RNA polymerase (transcription termination sequence). In the present invention, the four types of D
It is preferable to use a template DNA in which NA sequences are linked in this order and the total is 50% or more of the total DNA sequence.
If the total of the four DNA sequences is less than 50% of the total DNA sequence, it is necessary to synthesize and bind DNA sequences not directly related to protein synthesis, in addition to the DNA portion necessary for protein synthesis, It is costly and reduces the efficiency of protein synthesis. Examples of the cell-free extract used in the present invention include a wheat germ extract, an Escherichia coli cell extract, and a rabbit reticulocyte extract.

Hereinafter, each step from the preparation of the cell-free extract used in the present invention to the measurement of the protein synthesis activity in the cell-free protein synthesis system will be described in detail. i. Preparation of cell-free extract Preparation of cell-free extract differs depending on the material used,
Any ordinary method may be used. ii. Cell-free protein synthesis reaction In addition to the cell-free extract, the reaction solution contains DNA, RNA, RNA polymerase, the constituent amino acids of the protein, a buffer, an energy source such as ATP and GTP, creatine phosphate, ATP regeneration systems such as creatine phosphokinase, phosphoenolpyruvate and pyruvate kinase, dithiothreitol (D
TT), stabilizers such as spermine and spermidine, RN
Add an appropriate amount of ase inhibitor. The reaction is carried out at an optimum temperature depending on the cell-free extract used, the type of the target protein, and the like, and generally 20 to 40 ° C is appropriate.

Information on the amino acid sequence used for protein synthesis is provided to the protein synthesis system in the form of mRNA. This mRNA is synthesized from the template DNA by a transcription reaction using RNA polymerase. RNA polymerase
First, a promoter sequence specific to the type is found on the DNA and binds to that portion, and then RNA synthesis is started. RNA synthesis terminates spontaneously at the point where the DNA sequence is lost (run-off), or terminates when a terminator sequence specific to the type of RNA polymerase appears. Plasmid DN which has been mainly used in the past
In the method using A as a template, run-off
There was no difference in the efficiency of protein synthesis between the method and the method using the terminator sequence. For this reason, a simpler run
The -off method has been mainly used.

However, when a PCR product DNA was used as a template, a phenomenon was observed in which the efficiency of protein synthesis using the run-off method was inferior to that when a plasmid was used. For this reason, the cause was analyzed and research was advanced to improve the efficiency of protein synthesis. As a result, a terminator DNA sequence or a mutant terminator DNA sequence is ligated after (at the 3 ′ end) the sequence of the template DNA encoding the target protein, and the promoter DN
A protein is produced by using a template DNA in which the total of the A sequence, the ribosome binding DNA sequence, the DNA sequence encoding the target protein, and the terminator DNA sequence or the mutant terminator DNA sequence is 50% or more of the total DNA sequence. It was confirmed that the synthesis activity was significantly increased. Although the cause of the increase in the amount of protein synthesis (protein synthesis activity) in the present invention has not been elucidated yet, the mRNA produced by RNA polymerase is stabilized, the amount of mRNA synthesis is increased,
Presumed causes include that the higher-order structure of the RNA functions effectively in the protein synthesis system.

[0016] The RNA polymerase used in the present invention may have any commonly used sequence. However, considering the activity in a protein synthesis reaction solution and the availability of RNA polymerase itself, T7, T3, S3
An RNA polymerase derived from a virus such as P6 is desirable. Further, the terminator DNA sequence or the mutant terminator DNA sequence used in the present invention stops the transcription reaction of RNA polymerase, and converts the synthesized RNA into DNA.
Any DN having a function of releasing from
The A sequence can also be used. Usually, the RN used for the transcription reaction
It is desirable to use a terminator DNA sequence that is unique to A polymerase. However, substitution of some bases of the terminator DNA sequence specific to RNA polymerase,
A DNA sequence that has been deleted or inserted, which stops the transcription reaction of RNA polymerase, and
Any DN having a function of releasing from
It is clear that the A sequence can also be used in the present invention, and such a terminator DNA sequence is referred to herein as a “mutant terminator DNA sequence”.

The present invention further provides the above-described nucleic acid amplification method of the present invention, wherein the method is separately performed on at least two kinds of nucleic acid molecules, and each of the amplified at least two kinds of nucleic acids is used as a template. And a method for constructing a protein library consisting of at least two types of proteins respectively encoded by the obtained at least two types of nucleic acids. By performing the amplification method and the protein production method of the present invention on a plurality of types of target nucleic acid molecules, a protein library containing a desired protein can be simply and efficiently constructed.

Hereinafter, the present invention will be specifically described with reference to Comparative Examples and Examples. Example 1 (Amplification of Nucleic Acid) Preparation of Nucleic Acid Molecules for Amplification Plasmid pRSET-GFP1 shown in FIG.
PCR was performed under the following conditions using a DNA fragment obtained by digestion with aI as a template. The primer R2-0 used here had N (mixture of A, G, C, and T) at three places.
Wherein the 4 ³ types as the type of molecule, i.e. 64 types of D
It is a mixture containing NA molecules.

[0019]

Table 1 Final concentration of template DNA in reaction solution 2 ng / ml of template DNA Primer F2-1 1000 nM Primer R2-0 1000 nM dNTPs 0.2 mM Taq DNA polymerase 40 U / ml (TaKaRa) in total 50 μl dNTPs: deoxynucleotide triphosphate (four kinds of A mixture of bases)

Preheat at 94 ° C. for 3 minutes.
5 seconds -55 ° C, 30 seconds -72 ° C, 1 minute cycle 30
After cycling, it was further extended at 72 ° C. for 10 minutes. The reaction solution was subjected to phenol / chloroform treatment and ethanol precipitation treatment, which was further purified by HPLC under the following conditions. Column: TOSOH TSKgel DEAE-NPR Temperature: 25 ° C. Eluate A. 20 mM Tris-HCl (pH 9.0) 20 mM Tris-HCl (pH 9.0) +1.0 M N
aCl 0 minutes (B: 0%), 10 minutes (B: 50%), 30 minutes (B: 70%)
%) And 50 minutes (B: 100%). Flow velocity is 0.
5 ml / min, detection was at 260 nm. The collected fraction was subjected to phenol / chloroform treatment and ethanol precipitation treatment, and then dissolved in TE buffer. In order to eliminate double-stranded DNA other than the nucleic acid molecule group containing the target nucleic acid molecule, PCR was further performed under the following conditions.

[0021]

[Table 2] Final concentration of template DNA in reaction solution 5 μg / ml Primer F2-1 1000 nM Primer R2-1 1000 nM dNTPs 0.2 mM Taq DNA polymerase 40 U / ml (TaKaRa) total volume 50 μl

Preheat at 94 ° C for 3 minutes.
After 5 cycles of 5 seconds -53 ° C, 30 seconds -72 ° C, and 1 minute, elongation was further performed at 72 ° C for 10 minutes. The product of this reaction was purified by HPLC in the same manner as above to prepare a nucleic acid molecule group for amplification.

Dilution of Nucleic Acid Molecules Containing Target Nucleic Acid Molecules The HPLC purified DNA was diluted. At this time, to prevent DNA loss due to adsorption to a tube or the like, 10 ¹² molecules /
ml of the solution were sequentially added to 1.0 μg / ml tRNA (Sigma
It was diluted with a), to obtain a solution of 10 ³ molecules / ml. From this, 1 µl was taken and used for PCR.

Amplification of Single Kind of Molecules PCR was carried out under the following conditions using nucleic acid molecules in the diluted solution as templates.

[0025]

[Table 3] Final concentration of template DNA in reaction solution Diluted sufficiently and considered to be a single type of molecule. Primer F2-1 100 nM Primer R2-1 100 nM dNTPs 0.2 mM Taq DNA polymerase 40 U / ml (TaKaRa) total volume 10 μl

Preheat at 94 ° C for 3 minutes.
5 sec-53 ° C, 30 sec-72 ° C, 1 minute cycle of 50
After cycling, it was further extended at 72 ° C. for 10 minutes. Using the nucleic acid molecules in the reaction solution as a template, PCR was further performed under the following conditions.

[0027]

Table 4 Final concentration in the reaction solution 1 μl of the above PCR reaction solution Primer F2-2 500 nM Primer R2-2 500 nM dNTPs 0.2 mM Taq DNA polymerase 40 U / ml (TaKaRa) in total 11 μl Preheating at 94 ° C. for 3 minutes, 96 ° C, 15 seconds -58
C., 30 seconds-72.degree. C., 1 minute cycle was performed 30 times, followed by further extension at 72.degree. C. for 10 minutes. The reaction product was analyzed by agarose electrophoresis, and it was confirmed that a DNA band of a desired length was generated.

Comparative Example 1 The same operation as in Example 1 was performed. However, the purification by HPLC was not performed, and the concentration of the primers F2-1 and R2-1 was 1 μM in the PCR at the stage of amplification of a single type of molecule. The base sequences of the primers used are shown below.

[0029]

[Table 5] Primer for PCR Sequence F2-1 5 'GCGAGTCAGT GAGCGAGGAA G 3' F2-2 5 'CGATTCATTA ATGCAGATCT CGATCCC 3' R2-0 5 'AACAGTACAC GTCGTGCCAC CAAAGCAGAG AGCTCCACTG TCTCGCCAAN NNGATCAAGC TTCGAATTAC ACGAATGCTAG ATCACGATCAG C 3 ' R2-2 5' AGCAGAGAGC TCCACTGTCT CGCC 3 '

The sequence of the NNN portion of the clone obtained in Example 1 and the number of clones are shown in the following table.

[0031]

[Table 6]

The DNA sequence obtained from the final PCR reaction solution was a single sequence, and of the 64 sequences expected,
24 clones were obtained. In contrast, DNA obtained from the final PCR reaction solution of Comparative Example 1 was analyzed by agarose electrophoresis, but no DNA band of the desired length was detected. As described above, in the present invention, after isolating and purifying a nucleic acid molecule group containing a nucleic acid molecule to be amplified,
The amplifiable nucleic acid molecules other than the primers in the PCR reaction solution are made up of only the target nucleic acid molecule, and the concentration of the primer to be used is 10 per type of primer.
Since PCR is performed at 0 nM or less, a single nucleic acid molecule can be simply and efficiently amplified.

Example 2 (Synthesis of protein) Preparation of Escherichia coli extract (Ellman et al., Methods Enzymol. 202: 301-336 (199)
According to 1)), an E. coli extract was prepared. First, E. coli A
19 in a jar fermenter containing 8 L of culture solution
The culture was stopped at a temperature of 450 ° C. when the absorbance at 450 nm reached 1.1. The cells were collected by centrifugation, suspended in a buffer, and disrupted with a French press at 8400 psi.
Immediately after centrifugation, a 30,000 × g centrifuged supernatant (S30) was obtained.
A buffer containing an energy source such as ATP was added to the obtained supernatant, and the mixture was slowly shaken at 37 ° C. for 80 minutes. Subsequently, this solution was dialyzed three times at 4 ° C. for 45 minutes, and the supernatant was further centrifuged at 4,000 × g to obtain an E. coli extract.

Cell-Free Protein Synthesis Reaction Cell-free protein synthesis was carried out according to the method developed by Toshiya Endo et al. (69th Annual Meeting of the Japanese Biochemical Society, Publication No. 4-P-1209). The reaction solution of Example 2 was 56.4 mM Tris ·
HCl buffer pH 7.4, 1.22 mM ATP, 0.85 mM GTP each, UTP, CTP, 1.76 mM DTT, 40 mM creatine phosphate, 0.15 mg / ml creatine kinase, 320 μM amino acid, 10 mM magnesium acetate, 150 mM potassium acetate,
4% PEG, 34.6 μgl / ml folinic acid, 35.9 mM NH ₄ OAc,
10 μg / ml rifampicin, 0.17 mg / ml E. coli tRNA,
It consisted of 0.02 mg / ml template DNA, 10 μg / ml T7 RNA polymerase, and 0.2 ml of E. coli extract, and the total amount was 1 ml. The reaction was performed at 37 ° C. for 2 hours.

However, the template DNA used in Example 2 was T
7 promoter sequence, GFP (green fluorescein prot
ein: jellyfish fluorescent protein) Using the plasmid pRSET-GFP1 containing the gene sequence and T7 terminator sequence as a template,
Sense primers P1 and T containing T7 promoter sequence
It was obtained by performing PCR using an antisense primer P3 having a sequence further behind the 7 terminator sequence. In addition, plasmid pRSET-GFP1 is used for plasmid pGFP (Clont
ech) with the restriction enzymes BamHI and EcoRI to excise the GFP gene, which is then plasmid pRSETB (Invitrogen
Manufactured by BamHI and EcoRI. FIG. 2 shows the DNA structure of the portion of the DNA sequence of plasmid pRSET-GFP1 used as a template in this example. The T7 promoter sequence and ribosome binding DNA sequence used here are as follows. T7 promoter sequence TAATACGACTCACTATA ATTATGCTGAGTGATAT Ribosome binding DNA sequence AAGGAG TTCCTC

Example 3 (Synthesis of Protein) The reaction of Example 3 was carried out using a reaction solution having the same composition as that of Example 2. The template DNA was the same plasmid as in Example 2, and a short template DN obtained by performing PCR using a sense primer P1 containing a T7 promoter sequence and an antisense primer P2 immediately before the T7 terminator sequence.
A was used. One hour after the reaction, a portion of the reaction solution was
The amount of synthesized P was determined by measuring the fluorescence emitted by GFP, the excitation at 395 nm, and the emission at 509 nm. The amount of the synthesized protein is represented by a value obtained by subtracting the background fluorescence value at the reaction time 0 from the fluorescence value emitted from the GFP protein synthesized after a certain period of time.

Examples 4 and 5 (Synthesis of protein) In order to confirm the effectiveness of the terminator sequence shown in Example 2, the lengths of the template DNAs were adjusted.
The template DNA having the T7 terminator sequence was prepared in Example 5
Then, a template DNA in which a part (6 bases) of the T7 terminator sequence was changed was used. Preparation of E. coli extract and cell-free protein synthesis reaction Preparation of E. coli extract and cell-free protein synthesis reaction were performed in the same manner as in Example 2. However, the template DNA in Example 4 was obtained by performing PCR using the same plasmid as in Example 2 as a template and a sense primer P1 containing a T7 promoter sequence and an antisense primer P4 containing a T7 terminator sequence. The template DNA used in Example 5 was
A sense primer P1 containing a T7 promoter sequence;
It was obtained by performing PCR using an antisense primer P5 containing a sequence in which a part (6 bases) of the T7 terminator sequence was changed. The nucleotide sequences of the primers used for PCR are shown below.

[0038]

[Table 7] Primer base sequence P1 CCGCGAAATT AATACGACTC P2 TTATTGCTCA GCGGTGGCAG P3 GCCAGATCCG GATATAGTTC CTCC P4 AGCAAAAAAC CCCTCAAGAC C P5 AGCAAAAATC GTCTCAACAA GCGTTTAGAG GCCCCAAGGG GT The results are shown in Table 8 below.

[0039]

Table 8 Example GFP synthesis amount (relative value) Example 2 (with full-length T7 terminator connected) 15 Example 3 (without terminator connected) 2.2 Example 4 (with full-length T7 terminator connected) 13 Examples 5 (Partial change in sequence of full-length T7 terminator) 4.3

The protein (G) of Example 2 using a template DNA linked to a full-length T7 terminator DNA sequence
FP) It can be seen that the amount of synthesis is 6.8 times the amount of protein synthesized in Example 3 using the template DNA not linked to the T7 terminator DNA sequence. Also, full length T7
The amount of protein (GFP) synthesized in Example 4 using the template DNA linked to the terminator DNA sequence was 5.9 times the amount of protein synthesized in Example 3 using the template DNA not linked to the T7 terminator DNA sequence. I understand.

Further, the terminator DNA of Example 4
The amount of protein synthesized in Example 5 using a template DNA having the same length as the sequence and having a part of the sequence changed (mutant terminator DNA sequence) was lower than that in Example 4, although the amount of protein synthesis was lower. It can be seen that the amount of protein synthesis in Example 3 using a template DNA not linked to the T7 terminator DNA sequence was twice as large. The above results show that the use of a template DNA linked to a full-length terminator DNA sequence significantly increases the amount of protein synthesis, and that when a mutant terminator DNA sequence in which a partial sequence of the terminator DNA sequence is changed is used. This shows that the effect obtained by linking the terminator DNA sequence is reduced.

Examples 6 and 7 (Synthesis of protein) In this example, the plasmid pRSET-
PCR was performed under the same conditions as in Example 1 using a DNA fragment obtained by cleaving GFP1 with ScaI as a template. Of the DNA finally obtained using primers F2-2 and R2-2, the sequence of NNN was AAC
The following experiment was performed using the following.

Preparation of T7 terminator sequence PCR was carried out under the same conditions as in Example 1 using a DNA fragment obtained by cutting plasmid pRSET-GFP1 with ScaI as a template, and T7 capable of binding to the above sequence was obtained.
A terminator sequence was produced.

[0044]

[Table 9] Final concentration of template DNA in reaction solution 1 ng / 50 μl Primer F-T7TER-1 1000 nM Primer pRSET-13-32 1000 nM dNTPs 0.2 mM Taq DNA polymerase 40 U / ml (TaKaRa) total volume 50 μl

Preheat at 94 ° C for 3 minutes.
3 cycles of 5 seconds -55 ° C, 30 seconds -72 ° C, 30 seconds
After 0 cycles, extension was performed at 72 ° C. for 10 minutes.
The obtained DNA fragment was purified by HPLC in the same manner as in the example to obtain a T7 terminator fragment. Then this T
The 7 terminator fragment was added to the DNA fragment obtained by PCR of one molecule.

[0046]

Table 10 DNA obtained by PCR at a final concentration of 1 molecule in the reaction solution , 1 ng / 50 μl NNN sequence is AAC T7 terminator fragment 1 ng / 50 μl Primer F2-2 500 nM Primer pRSET-13-32 500 nM dNTPs 0.2 mM Taq DNA polymerase 40 U / ml (TaKaRa) Taq Start Antibody (Clonetech) 1.76 ng / ml Total volume 10 μl

Preheat at 94 ° C. for 3 minutes.
5 seconds at -58 ° C, 30 seconds at -72 ° C, 1 minute cycle of 30
After cycling, it was further extended at 72 ° C. for 10 minutes. P
The base sequence of the primer used for CR is shown below.

[0048]

[Table 11] Primer Base sequence F-T7TER-1 GGCGAGACAG TGGAGCTCTC TGCCCGGCTG CTAACAAAGC C pRSET-13-32 CTGCGCAACT GTTGGGAAGG G

Next, an E. coli extract was prepared and a cell-free protein synthesis reaction was carried out in the same manner as in Example 2. The results are shown in Table 11 below.

[0050]

Example 12 GFP synthesis amount (relative value) Example 6 (with full-length T7 terminator connected) 3.6 Example 7 (without terminator connected) 0.79

The above results indicate that the full-length terminator DN
This shows that the use of the template DNA linked with the A sequence significantly increases the amount of protein synthesis.

[0052]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid PCR primer Sequence GCGAGTCAGT GAGCGAGGAA G 21 SEQ ID NO: 2 Sequence length : 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid PCR primer Sequence CGATTCATTA ATGCAGATCT CGATCCC 27 SEQ ID NO: 3 Sequence length: 80 Sequence type: Nucleic acid chain Number: single-stranded Topology: linear Sequence type: other nucleic acid PCR primer sequence AACAGTACAC GTCGTGCCAC CAAAGCAGAG AGCTCCACTG TCTCGCCAAN NNGATCAAGC 60 TTCGAATTCT ACGAATGCTA 80 SEQ ID NO: 4 Sequence length: 21 Sequence type: Nucleic acid Number of strands : Single strand Topology: Linear Sequence type: Other nucleic acid PCR primer Sequence AACAGTACAC GTCGTGCCAC C 21 SEQ ID NO: 5 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Straight Type Sequence type: Other nucleic acid Primer for PCR Sequence AGCAGAGAGC TCCACTGTCT CGCC 24 SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid PCR primer sequence CCGCGAAATT AATACGACTC 20 SEQ ID NO: 7 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid PCR primer sequence TTATTGCTCA GCGGTGGCAG 20 SEQ ID NO: : 8 Sequence length: 24 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid PCR primer Sequence GCCAGATCCG GATATAGTTC CTCC 24 Sequence number: 9 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid PCR primer Sequence AGCAAAAAAC CCCTCAAGAC C 21 SEQ ID NO: 10 Sequence length: 42 Sequence type: Number of nucleic acid strand : One Topology: Linear Sequence type: Other nucleic acid PCR primer Sequence AGCAAAAATC GTCTCAACAA GCGTTTAGAG GCCCCAAGGG GT 42 SEQ ID NO: 11 Sequence length: 41 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence Type: Other nucleic acid PCR primer Sequence GGCGAGACAG TGGAGCTCTC TGCCCGGCTG CTAACAAAGC C 41 SEQ ID NO: 12 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids PCR primer sequence CTGCGCAACT GTTGGGAAGG G 21

[Brief description of the drawings]

FIG. 1 is a drawing showing fragment DNA obtained by digesting plasmid pRSET-GFP1 with ScaI used as a template in Example 1, and the position of each primer.

FIG. 2 shows the DNA sequence of plasmid pRSET-GFP1.
7 shows a DNA sequence of a portion used as a template in Example 2.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Reiko Okumura 214 Tokiwa Mansion 205, Miyato-cho, Showa-ku, Nagoya-shi, Aichi Prefecture

Claims (10)

[Claims] In a method for amplifying a target nucleic acid molecule by PCR, after isolating and purifying a nucleic acid molecule group containing a nucleic acid molecule to be amplified, an amplifiable nucleic acid molecule other than a primer in a PCR reaction solution is converted to a target nucleic acid molecule. A method for amplifying a nucleic acid molecule, characterized in that PCR is carried out in a state consisting only of a nucleic acid molecule and the concentration of a primer to be used is set to 100 nM or less for one kind of primer. 2. A nucleic acid molecule group containing a nucleic acid molecule to be amplified is isolated and purified, and then diluted, so that the amplifiable nucleic acid molecule other than the primer in the PCR reaction solution is composed of only the target nucleic acid molecule. The method of claim 1, wherein 3. The method according to claim 1, wherein the nucleic acid molecules are isolated and purified by HPLC. 4. The method according to claim 1, wherein the concentration of the primer in the PCR reaction solution is 10 nM or less per one kind of primer. 5. The method according to claim 1, wherein nested-PCR is used.
The described method. 6. A method for producing a protein in a cell-free protein synthesis system containing a cell-free extract, wherein a single kind of nucleic acid obtained by the method according to any one of claims 1 to 5 is used as a template. A method comprising: 7. The nucleic acid used as a template is a promoter DNA sequence, a ribosome binding DNA sequence, a DNA sequence encoding a target protein, and a terminator DNA.
The method according to claim 6, which is a template DNA containing a sequence or a mutant terminator DNA sequence, and wherein the total of the four DNA sequences is 50% or more of the total DNA sequence. 8. The RNA polymerase used for transcription is T7
The method of claim 7, wherein 9. The method according to claim 6, wherein the cell-free extract is an Escherichia coli extract. 10. The method according to any one of claims 1 to 5, wherein the method is separately performed on at least two kinds of nucleic acid molecules, and each one of the amplified at least two kinds of nucleic acids is used as a template. 10. A method for carrying out the method according to any one of claims 6 to 9 for each nucleic acid, and constructing a protein library comprising at least two types of proteins respectively encoded by the obtained at least two types of nucleic acids.