JPS60104097A - Purification of dna - Google Patents

Abstract

PURPOSE:To purify DNA in a short time efficiently, by bringing an aqueous solution containing DNA and mononucleotide into contact with an adsorbent previously swelled, shaving a hydrophilic stationary phase on the surface, adsorbing and separating DNA, eluting it with an eluting solution such as methanol, etc. CONSTITUTION:An aqueous solution containing DNA and mononucleotide is brought into contact with an adsorbent previously swelled, having a hydrophilic stationary phase on the surface, DNA is adsorbed on a stationary phase, the adsorbent having adsorbed DNA is separated from the aqueous solution containing mononucleotide, the adsorbent is brought into contact with an eluting solution consisting of methanol or an aqueous eluting solution containing a polar organic solvent, DNA is eluted in the eluate, so DNA is purified. DNA is preferably that which is phosphorylated at 5'-end, and mononucletide is preferably adenosine triphosphate and/or adenosine diphosphate.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying DNA, and more particularly to a method for purifying DNA from a solution containing DNA and mononucleotides using an adsorbent.

As genetic recombination technology has developed in recent years, the usefulness of DNA obtained through chemical synthesis and the like has increased. ':50-
As a primer, for the improvement of overlapping Otter, etc., we have further developed a structural gene of several hundred and thousands of bases - LP''.
5 DNA, which serves as the synthetic unit of the lasmid gene, has started to play an important role.

Such NA often becomes a mixed solution with a mononucleotide such as adenosysinosic acid during its preparation or actual use. For example, when such NA is actually used, its 5' end is enzymatically phosphorylated using labeled or unlabeled adenosysyltrisic acid (hereinafter abbreviated as ATP). There are many things. Usually, an excess amount of ATP is used for this phosphoric acid esterification, but after the reaction, the target phosphoric acid esterified DNA is separated and purified from the excess ATP and the adinosine dirisic acid (hereinafter abbreviated as ADP) generated in the reaction. There is a need. Furthermore, since the polystyrenease used in the reaction often inhibits the next reaction, it is desirable to remove it. It is an important issue to efficiently separate and purify DNA from a solution containing t-nonucleotides.

Gel filtration, dialysis, iosiexchange, and ethanol precipitation are generally used to remove ATP 9 ADP. Ethanol precipitation is mainly used especially for processing small amounts of DNA. Furthermore, phenol treatment is mainly used to remove enzymes. However, these methods are complicated and take a long time. In particular, in the case of DNA oligomers, it is necessary to use a large amount of t-RNA, etc., and removal of phenol after deproteinization is troublesome.
child.

On the other hand, it is known that DNA whose 5' end has been phosphated can be captured from an aqueous solution on a lipophilic stationary phase of particles having a lipophilic stationary phase and desalted. However, such operations have so far been performed after removing ATP and ADP, which are heptanonucleotides, and the 5' end of the DNA has been phosphorylated and ATP has been removed.
The behavior of each nucleotide in the presence of Jp ADP was not known.

As a result of a deep study on the behavior of nucleotides on a lipophilic stationary phase, the present inventors found that 5'-terminally phosphoric acid esterified DNA is captured from an aqueous solution onto a lipophilic stationary phase, but 8TP and ADP Surprisingly, heptanoscleotides such as E. coli were not captured by this, and the enzyme protein used for 5'-terminal phosphorylation was captured on the lipophilic stationary phase, but DNA
The present invention was completed by discovering that it is not desorbed from a lipophilic stationary phase during elution.

That is, in the present invention, an aqueous solution containing DNA and heptanonucleotides is brought into contact with a pre-swelled adsorbent having a lipophilic stationary phase on the surface, and LINA is absorbed into the lipophilic stationary phase, and this DNA is adsorbed. The method is characterized in that the adsorbent is used to separate an aqueous solution containing the nonucleotides, and the DNA is further contacted with an eluate consisting of methanol or an aqueous eluate containing a polar organic solvent to elute the DNA into the aqueous eluate. The present invention provides a method for purifying DNA.

The present invention provides 1) tlA in which the 5' end is esterified.
It is preferably applied when the product #1-.

The 5'-end phosphoric acid esterified DNA that can be purified in the present invention is of course an oligonucleotide or polynucleotide having a dinucleotide or more, preferably a tetranucleotide or more. Moreover, there is no particular limitation on the upper limit. Its base components are adenine (abbreviated as A),
It may be any of Chimi, 7 (abbreviated as T), Taani, 7 (abbreviated as G), and Shitoshishi (abbreviated as C).

Further, the phosphoric acid at the 5' end may be labeled with 32p or the like. The coexisting seven nonuku V otide also [γ-32P]
It may be labeled like ATP. There are no special restrictions regarding the concentration of these components in the aqueous solution to be purified.I) The 5' end of NA is converted to ATP in the presence of polyscleotide + an enzyme such as niacetyl ester. It may be in a diluted or concentrated state as required.

There is no particular limitation on the concentration of salts or the like in the aqueous solution to be purified, and the concentration may be sufficient to make up the entire buffer solution when performing the above-mentioned phosphoric acid esterification. However, it may also be possible to have a lower salt concentration or the absence of salts, or a higher salt concentration, for example on the order of several seven salts. The pH of the liquid needs to be within a range that does not cause denaturation of the DNA to be purified, but it may be at a pH that allows for the above-mentioned phosphoric acid esterification. Usually about pH 5 to about 9, preferably pH
It is around 7.

In the aqueous solution to be purified, polyscleotide +
It may also contain an enzyme protein such as N-7.

The adsorbent having a lipophilic stationary phase on its surface used in the method of the present invention includes particles of cross-linked organic polymers such as cross-linked bunbun, acrylic polymers, and styrene polymers, and particles of inorganic polymers such as solid silicic acid. An example of an adsorbent having a hydrophobic group such as a hydrocarbon group on the surface, which is a so-called carrier for reversed phase chromatography, can be mentioned.

In the present invention, such an adsorbent is used in a swollen state. This swelling may be carried out in a conventional manner, for example by soaking such adsorbents in a hydrophilic organic solvent such as methanol, or by passing such a solvent through a column packed with these adsorbents. It can be done easily.

The aqueous solution containing 5'-terminally phosphoric acid-esterified DliA and heptadonucleonade may be brought into contact with these adsorbents by a batch method or a column method according to a conventional method.

Separation of the adsorbent adsorbed with 5'-terminally phosphoric acid esterified DNA from the aqueous solution containing the mononucleotide is achieved by washing the adsorbent with water or an aqueous solution and separating the washing solution. can do. The aqueous solution used for removal has a pH of about 5 to about 9, preferably about 7, and a buffer concentration of O to about IM, preferably about 0.01M. The buffers used are triethylamide diacetate (hereinafter abbreviated as TEAA) and triethylamishiba carbonate (hereinafter abbreviated as TEA).
Particularly preferred are volatile buffers such as B).

The adsorbent thus separated from the aqueous solution containing mononucleates is further contacted with an eluate consisting of methanol or an aqueous eluate containing a polar organic solvent. The polar organic solvents constituting this eluate include lower alcohols such as methanol, ethanol, and ethyrecitalicon, hydrophilic ethers such as tetrahydrofuran and ethyrecitlycol 7-methyl ether, ketones such as acetyl alcohol, etc. Other non-protocytic polar organic solvents such as human ditolyl may be exemplified.

These hydrophilic organic solvents are used in the form of a mixed solution with water. The mixing ratio of water and hydrophilic organic solvent may vary depending on the type of hydrophilic organic solvent. For example, in the case of methanol, which has relatively strong polarity, it ranges from 100% methanol to about 1 part by weight of methanol per 1 part by weight of water, and with arc-tocitrile, which has relatively weak polarity, it ranges from about 0% methanol to 1 part by weight of water. The amount is about 15 parts by weight to about 0.6 parts by weight.

This aqueous eluate preferably further contains a buffer.

Any conventional buffering agent may be used, but it may be added later by azeotropy.
C-removable slow machining, such as TEAA, TEAB, etc., is particularly preferred. Its concentration, liquid properties, etc. may be the same as those for a single batch used in this technical field, and may be similar to those of an aqueous solution used for washing the adsorbent. The amount of the eluate used is about twice or more, preferably about three times or more, the amount of smoked sea bream in terms of ratio. There is no particular limit on the upper limit.

Since there is a delay in elution due to the washing solution remaining in the adsorbent, purified DNA can be obtained at a considerably high a degree by collecting both necessary fractions using a fraction collector. The concentration of DNA in the eluate can be within a range allowed by the solubility of DNA, for example, usually up to about 2 times.

Processing in each process mentioned above? Objective D for m fL
There are no particular limitations as long as denaturation of NA, etc., boiling of the solvent, freezing, etc. are not caused, but all the steps can be carried out at room temperature without causing any problems.

According to the method of the present invention, DNA can be separated not only from mononucleotides but also from contaminating proteins. For example, in the method of the present invention, the 5' end of the DNA is polynucleotide, the phosphoric acid at the γ-position is phosphorylated with labeled ATP in the presence of otide+nase, and then the phosphorylated DNA is It is particularly suitable for separation and purification from ATP, etc. In such cases, polynucleotide + enzyme is captured together with DNA on a lipophilic stationary phase,
Not eluted during the arrow elution process.

The method of the present invention can also simultaneously achieve zero desalting of DNA. That is, by treating a buffer solution containing DNA and heptanoscleotide according to the method of the present invention, eluating it with an eluent containing a volatile buffer, and removing the volatile buffer from the eluate by distillation, it is easy to obtain a salt-free solution. DNA is obtained.

As is clear from the above description, according to the method of the present invention, DI particles that have leaked Nisdell ricin acid can be removed by gel filtration, dialysis,
It can be easily separated and purified from heptanonucleotides, proteins, salts, etc. without complicated steps such as ion father exchange and ethanol precipitation.

The present invention will now be celebrated in more detail by way of examples.

Example 1 5'HO old jmer (15mer, Ho(JTAAA)
GTGTCCGAG"') Aqueous solution containing 0,12 pt μz, [r -32P) Aqueous solution containing ATP10 micro + Yuri (ATP amount 0.5X10 7L)
μL and T4-polyscleonate agent! (Manufactured by Takara Shuzo Co., Ltd.) Approximately 1 μL of Mizunuma liquid containing 3 units
507 tris(human O+dimethyl)aminomethane, 5
0ml magnesium chloride, 10'rrML spermine, 50rrMi dithiothreitol and 0.5M
It was mixed with 1 μt of a pH 9.6 buffer solution containing potassium chloride, and diluted to 5 μHc by adding water.

This solution was maintained at 37° C. for 1 hour to perform caination of the 5′ HO oligomer. After the caination reaction, the reaction solution was diluted with water to make 50 μL.

On the other hand, the bottom end of a column with an inner diameter of 4 wire and a diameter of 4.5 cm was plugged with absorbent cotton, and anhydrous silicic acid *1 ~ C was added to the particle surface.
ig hydrocarbon group-containing carrier gel for reverse-phase O matocorafish (particle size 32-48μ, manufactured by Waters, 0-18
gel) was filled to a height of approximately 1 crn. 1 in this column
ml of methanol to swell the gel, and then add triethylamine acetate buffer (pH 7.0,
Conditioning was performed by flowing 3 mA (1M).

40 .mu.t of the diluted reaction solution obtained in the previous Kaineshoshi reaction was applied to the column thus prepared, and the column was further washed with triethyla'E and cyacetate buffer (0.1M) 3- of pH 7.0.

Next, an eluent prepared by mixing 7 parts by volume of a triethylamine acetate buffer (0.1 M) with a pH of 7.0 and 3 parts by volume of acetonitrile was passed through the column, and the effluent was collected.

The eluate and diluted reaction solution thus obtained were spread as a thin layer and developed using zero-magnetography.Carrier: DEAE cell O-su, developer: t
- RNA degradation H73xure (containing 7M urea) and autoradiolysis were performed. Is the diluted reaction product glue O madokram 32p years old Rico? -(15mer), "2P-
A strong spot corresponding to ATP etc. was observed, but only a strong spot corresponding to 32p oligomer (15mer) was observed from the eluate. No protein was detected in the bath fluid.

Example 2 5'HO ori j? (20mer, HoAATOG
GGOAl['GGATTTCC1ji0'')0.6μV
The caination reaction and DNA purification were carried out in the same manner as in Example 1 using the following. However, instead of pH 7.0 triethylamine acetate buffer (0.1M), pH 7.0
Triethylamici hydrogen carbonate (0.1M) was used.

The obtained eluate was concentrated and dried under reduced pressure, and further diluted with water 10
0 .mu.t was added and dried under reduced pressure, and this was dissolved in 100 .mu.L of water to obtain an almost pure aqueous solution of 5'-32P oligomer. The recovery based on the 5'HO mer was almost quantitative.

Example 3 Example 20 Other 5'HO oligomer instead of 5'HO oligomer: j ? -(20mer,"'GCTGGGGCGA
Using AA demand AAGACTG0H), [γ-32P)
Example 2 was repeated using unlabeled ATPO, 5×10=73, in place of ATP. Similar to Example 2, almost pure 5'-P oligomer (AAAGG with weak PGC) was used.
An aqueous solution was obtained.

Example 4 Size marker DNA obtained by cutting pBR322 lasmid with restriction enzyme MboI (8, 11, 12
,15,17,18,27,31,36,46,75,
77, 91, 105, 207, 258, 272, 317
, 341, 358, 665 and 1374 base pairs) containing 0.1 pF was prepared from the 5'HO of Example 1.
Example 1
Caination was performed in the same manner.

Furthermore, DNA was purified according to Example 1.

However, the reaction product solution was not diluted, and 1 .mu.t of it was collected and used, and methanol 1.mu.t was used as the eluent instead of the mixed eluent of triethylamine acetate buffer and acetonitrile.

The eluate thus obtained was subjected to thin layer chromatography and autoradiochemistry in the same manner as in Example 1,
It was confirmed that [γ-32P] ATP was not substantially trapped in this.

This eluate was concentrated and dried under reduced pressure, and an 80% formamide aqueous solution (hereinafter referred to as dye (referred to as solution)5
0 μt was added and dissolved, and this was collected in 6 pt portions. The fractionated solution was heated to 90°C for about 2 minutes and then rapidly cooled to bind the double-stranded DNA into single-stranded DNA. Conduct electrophoresis (migration solution: pH 8.0 aqueous solution containing 0.1M 1-lis(hydro-dimethyl)aminomethane, 0.1M boric acid and 2mM EDTA. Potential gradient: 28 V/m), autoradio. It was displayed on X-ray film using Kuramui.

The pattern of the obtained autoradioclam is shown in Figure 1 (a photograph is substituted). Virtually all 32P DNA was recovered. The autoradiograms shown in FIGS. 1A and A' were obtained after purification of 32P DNA, and the autoradiogram shown in FIG. 1 was obtained from the reaction product solution for comparison. [γ-32P) ATP in the reaction product solution leaked into the electrophoresis solution during electrophoresis, and therefore did not appear in the autoradioclum.

Example 5 5'-32p oligomer obtained in the same manner as in Example 2 and 5'-P oligomer obtained in the same manner as in Example 3.
．． 6μ? and 5'-HO oligomer (12mer,
HoAAGGACCGACCC0H) Approximately 4.0p? 30mM MgGl-z8pt and 100mMN-
2-Diethyldihydride-N'-2-ethanesulfodiic acid 8ILt was dissolved in a slow* solution, and water was added to give 2.
It was set to 4μt.

This solution was heated to 70°C for 10 minutes, returned to room temperature, left for 2 hours, and cooled to 12°C for 1 hour. This is 207 AT
P aqueous solution 1 μt, 3007 cinnaothreitol aqueous solution 1 μt and T4DN 8 liquor e (% l unit,
0.6 units and 0.3 units) and heated to 12°C.
The reaction was allowed to occur overnight.

DNA was purified from the reaction product solution according to Example 1 to obtain a solution containing purified DNA. This solution was substantially free of ATP and ADP. The obtained purified DNA bath was dried under reduced pressure, dissolved in a dye solution in the same manner as in Example 4, and subjected to electrophoresis and autoradiochemistry. The pattern of the obtained autoradioclam is shown in Figure 2 (a photograph is substituted). A in Figure 2,
A' is an autoradiogram performed on the 2P DNA mixture obtained in Example 4 as a marker, B, C
and D are 1.0 units and 0 units on T4-liquor, respectively.
．． This is the autoradiocrum pattern of the sacsiple when using 6 units and 0.3 units. A gel portion corresponding to 40 smoke groups on the X-ray film was cut out, placed in a deposition film, and 0. IM tris(dimethyl)aminomethane, 0.1M boric acid and 2mM EDTA
Extraction was performed by electrodialysis in a pH 8.0 buffer containing . For electrodialysis, a voltage of 50 volts was applied overnight, and the solution in the dialysis membrane was collected after 10 minutes of reverse travel.

The same reversed-phase magnetic carrier used in Example 1 was used as a four-ball, swelled and squeezed in a column, and the obtained solution was passed through the column. Washing and elution was carried out in accordance with Example 1 below, and the obtained eluate was concentrated and dried under reduced pressure. The process of adding water to the dried product, concentrating it under reduced pressure, and drying it is repeated three times.Finally, the product is dissolved in water to obtain the desired 5' −
32p orij? -(20mer, 32P AATC
SakijiCATGGATTTOCTGGCT-GGGCGA
An aqueous solution of AAC; GAAGAC; TG) was obtained. The yield was almost quantitative.

[Brief explanation of drawings]

FIGS. 1 and 2 are photographs substituted for drawings showing autoradiograms of the electrophoretic pattern of DNA purified by the method of the present invention on a polyacrylamide denaturing gel. Procedural amendment (method) August 1932, Showa';'S-q:qν d1 application No. 7FD I-
No. 3 Relationship with famous event A'1 to be amended Application Person residence + Sui 2-16-2 Marunouchi, 111-ku, Cho-dai, Tokyo
Shigesu Building 3305 within the No. Marunouchi, IJ I of the amendment order 1939 'Z, If zg 43 ' -1lr-ke ga oki-ke- 7, the image of the amendment The following matters in the specification of the present application In Note 1, page 19, line 9, the phrase ``Drawing showing an autoradiogram'' is corrected to ``Drawing showing an autoradiogram (X-ray photograph).''

Claims (1)

[Claims] I. An aqueous solution containing DNA and mononucleotides is brought into contact with a pre-swollen adsorbent having a lipophilic stationary phase on its surface to adsorb the DNA to the stationary phase, and the adsorbent adsorbed with DNA is separated from an aqueous solution containing mononucleotides,
A method for purifying DNA, which further comprises contacting the DNA with an eluent comprising methanol or an aqueous eluent containing a polar organic solvent to elute the DNA into the eluate. 2 D where DNA is phosphorylated at its 5' end
NA, and the mononucleotide is adenosine trisic acid and/or adenosissinyl acid, Claim 1
Purification method described in section. 3. The purification method according to claim 1 or 2, wherein the adsorbent adsorbing D1'JA is separated from the aqueous solution containing the mononucleotide by washing the adsorbent with water or an aqueous solution.