JPH07227284A - Method for treating synthetic dna - Google Patents

Abstract

PURPOSE:To surely treat a synthetic DNA without waste by cutting off the synthetic DNA from a resin, diluting a deprotected DNA solution with water, passing the diluted DNA solution through a container packed with a porous carrier to make an oligonucleotide adsorbed, optically determining an adsorbed amount of the oligonucleotide and eluting the adsorbed substance. CONSTITUTION:A synthetic DNA is mixed with ammonia water, an oligonucleotide protected from a resin is cut off and a DNA solution obtained by deprotection is diluted with water. The diluted DNA solution is passed through a container packed with many porous carriers and the oligonucleotide is adsorbed and supported on the carrier. Then, the amount of the oligonucleotide adsorbed on the carrier is determined by an ultraviolet and visible light spectrophotometer to confirm sure adsorption and support of the oligonucleotide on the carrier. An acidic liquid is passed through the container to remove DMT group of the oligonucleotide. After cleaning, an organic solvent is passed through the container, the oligonucleotide is eluted from the carrier and recovered to surely treat without a mistake while preventing occurrence of waste before it happens.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for treating synthetic DNA for recovering an oligonucleotide by cleaving it from a resin.

[0002]

2. Description of the Related Art As a method for treating this kind of synthetic DNA,
Ammonia water is added to the synthetic DNA to separate the "protected oligonucleotide" from the resin, and the deprotected DNA solution is diluted with water, and the diluted solution is placed in a container containing many carriers. Through a second step of adsorbing and holding the oligonucleotide on the carrier, a third step of removing impurities by passing a washing solution into the container, and a fourth step of removing an DMT group of the oligonucleotide by passing an acidic solution into the container. , Through a fifth step of removing the acidic liquid by passing wash water through the container, and a sixth step of passing an organic solvent liquid through the container and eluting and collecting the oligonucleotide from the carrier in the container, It is known to recover oligonucleotides.

[0003]

The problem lies in the second step. When the DNA solution obtained in the first step is diluted by adding water and the diluted solution is passed through the container, the porous carrier in the container contains the oligonucleotide without being adsorbed and retained in the passing solution. May be discharged outside the container. In that case, the processes from the third step onward are all wasted work. Moreover, in the finally refined state, it is not even visually confirmed, and there is a problem in that the notice is delayed. In order to prevent the inconvenience, it is conceivable to store the passing liquid in the second step to the end as a second best measure and start again from the second step. However, this is also a great waste. Therefore, an object of the present invention is to make it possible to confirm whether or not the oligonucleotide is adsorbed and retained on the carrier in the container by a simple method but surely before moving to the third step through the second step, Accordingly, it is an object of the present invention to provide a method for treating synthetic DNA which can eliminate the waste of conventional treatment operations.

[0004]

The method for treating DNA according to the present invention comprises the following first to sixth steps. First Step Ammonia water is added to the synthetic DNA to separate the "protected oligonucleotide" from the resin. Second step The deprotected DNA solution is diluted with water, and the diluted solution is passed through a container containing a large number of porous carriers to adsorb and hold the oligonucleotide on the carriers. At this time, the amount of the oligonucleotide adsorbed on the carrier in the container is quantitatively analyzed by an ultraviolet-visible spectrophotometer. Third Step Impurities are removed by passing a cleaning liquid into the container. Fourth Step An acidic liquid such as TFA (trifluoroacetic acid) is passed through the container to remove the DMT group of the oligonucleotide. Fifth step: The washing liquid is passed through the container to remove the acidic liquid. Sixth Step An organic solvent solution such as acetonitrile or ethanol is passed through the container, and the oligonucleotide is eluted from the carrier in the container and collected. Alternatively, in the second step, instead of detecting the amount of the oligonucleotide adsorbed to the carrier in the container, the content of the oligonucleotide in the passage solution discharged from the container is quantitatively analyzed by an ultraviolet-visible spectrophotometer. To do. More specifically, after evaporating the organic solvent from the eluate obtained through the sixth step, purification is performed by high performance liquid chromatography. Alternatively, water is added to the eluate obtained through the sixth step to dilute it, and the diluted eluate is purified by high performance liquid chromatography.

[0005]

In the second step, the amount of the oligonucleotide adsorbed on the carrier in the container is quantitatively analyzed by a known UV-visible spectrophotometer. Step 3 The following steps are entered. In the same second step, the content of the oligonucleotide in the passage solution discharged from the container was quantitatively analyzed by a visible spectrophotometer, whereby the passage solution was not contaminated with the oligonucleotide and was necessary for the carrier. If it can be confirmed that a sufficient amount of oligonucleotide is adsorbed and retained, the procedure from the third step onward is started also in this case. In any case, when a predetermined amount of the oligonucleotide is not adsorbed and held on the carrier in the container, the operation of passing the passing solution through the container again is repeated, and the oligonucleotide is added to the carrier in the second step. Make sure to hold it by suction.

[0006]

【Example】

(Example 1) First step 30% aqueous ammonia is added to synthetic DNA, and the "protected oligonucleotide" is separated from the resin such as glass and polyethylene at room temperature for 2 hours, and at the same time, the protecting group is also removed. Second step The same volume of water is added to the DNA solution deprotected in the first step to dilute it, and the diluted solution is passed through a container containing a large number of porous C18 (carbon number 18) spherical carriers. Only the "deprotected oligonucleotide" is adsorbed and held on the carrier by applying pressure with an active gas. At this time, the amount of the oligonucleotide adsorbed to the carrier in the container is quantitatively analyzed by a known UV-visible spectrophotometer to confirm that the carrier adsorbs and holds the oligonucleotide. At this time,
When the oligonucleotide is not adsorbed and retained on the carrier in a necessary and sufficient manner, the passage liquid from the container is passed through the container again. Thus, it is confirmed at this stage that the predetermined amount of the oligonucleotide is surely adsorbed and held on the carrier.

Third step 3% ammonia water is repeatedly passed twice into the container containing the carrier to leave the oligonucleotide on the carrier, and other impurities are washed away. Step 4 2% TFA (trifluoroacetic acid) is passed through the same container to remove the DMT group of the synthetic DNA for elution. Fifth step Water is passed through the same container to wash and remove the TFA. Sixth step 20% acetonitrile as an organic solvent liquid is passed through the same container to elute the target oligonucleotide from the carrier out of the container, and the eluate is collected in a second container. Up to this point, processing is automated. Seventh Step Next, the eluate of the oligonucleotide is freeze-dried, and the organic solvent is evaporated. Eighth step Finally, the oligonucleotide obtained by freeze-drying is finally purified by high performance liquid chromatography.

(Example 2) In the second step of Example 1, the passing solution from the container containing the carrier was recovered, and the content of the oligonucleotide in the passing solution was quantitatively analyzed by an ultraviolet-visible spectrophotometer. Then, after confirming that the passing solution did not contain the oligonucleotide, that is, the oligonucleotide in the container was adsorbed and retained by the carrier in the necessary and sufficient manner, the process proceeded to the next third step. Also in this case, when the predetermined amount of the oligonucleotide is not adsorbed and held on the carrier in the container by the quantitative analysis, the passage liquid is re-passed into the container again. The other conditions were the same as in Example 1.

Example 3 In Example 1 or 2, after water was added to the eluate of the oligonucleotide obtained through the 6th step to dilute it, the 7th step of Example 1 was omitted. The diluted solution was purified by high performance liquid chromatography.

[0010]

EFFECTS OF THE INVENTION According to the present invention, it is confirmed in advance that the oligonucleotide is adsorbed and held on the carrier in the container in the second step by quantitative analysis with an ultraviolet-visible spectrophotometer before the third step. Since it is put in the box, there is no waste in the subsequent processing steps and it is efficient as a whole. Moreover,
Since only a known UV-visible spectrophotometer is used, no special cost is required, and the processing can be surely performed without error.

Claims (4)

[Claims] 1. A first step of adding ammonia water to the synthetic DNA to separate the "protected oligonucleotide" from the resin, and diluting the deprotected DNA solution with water to form a porous solution of the diluted solution. A second step of passing the carrier through a container containing a large number of carriers and adsorbing and holding the oligonucleotide on the carrier, a third process of passing a washing solution through the container to remove impurities, and an acidic solution passing through the container for DM of oligonucleotide
Fourth step of removing the T group, and fifth step of passing the washing water through the container to remove the acidic liquid
And a sixth step of passing an organic solvent solution through the container to elute and recover the oligonucleotide from the carrier in the container, and in the second step, the amount of the oligonucleotide adsorbed to the carrier in the container A method for treating synthetic DNA, which comprises quantitatively analyzing with a UV-visible spectrophotometer. 2. The method of treating synthetic DNA in the second step, which comprises quantitatively analyzing the content of the oligonucleotide in the flow-through solution discharged from the container with an ultraviolet-visible spectrophotometer. 3. A method for treating synthetic DNA, which comprises evaporating an organic solvent from the eluate obtained through the sixth step and then purifying it by high performance liquid chromatography. 4. A method for treating synthetic DNA, which comprises diluting an eluate obtained through the sixth step by adding water, and purifying the diluted eluate by high performance liquid chromatography.