JP2003245069A - Method for preserving dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for dry-preserving a DNA chemically boned onto a solid. <P>SOLUTION: The technique comprises rapidly drying the DNA, after chemically bonded onto the solid, by hot- or freeze-drying at a reduced pressure; wherein, (1) the hot-drying conditions are: 40-95°C and 4 Pa(4*10<SP>-6</SP>N/mm<SP>2</SP>), and (2) the freeze-drying conditions are: ≤0°C and a specified reduced pressure limit which can be determined according to the phase diagram for water and ice. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method in which DNA is chemically bonded to a solid such as artificial diamond, glass, silicon, graphite and various plastics and rapidly dried by vacuum heating drying and vacuum freeze drying. It is related to the saving technology. The reduced pressure heating and drying conditions are 4
Reduced pressure from 0 ° C to 95 ° C is 4 Pa (4 ^* 10 ^-6 N / mm
² ) It can be dried if it is below, and the freeze-drying condition is 0 ℃
Decompression limits can be determined according to the water and ice phase diagram if:

[0002]

2. Description of the Related Art A conventional DNA storage method is as follows. B) For storage of the DNA alone, sterilize water (sterilized by high temperature and high pressure), DIMSO (dimethyl sulfoxide aqueous solution), minus 3 when dissolved in a solvent such as alcohol.
Store in an environment below 0 ° C. B) Incorporate DNA into various vectors and store at -30 ° C
Save in the following environment. C) DNA is chemically bonded to the solid (hereinafter, DNA-immobilized chip: Japanese Patent Application No. 11-320945), and the temperature is -30 ° C.
Save in the following environment. D) The DNA-immobilized chip is a chip in which DNA is chemically bonded to a solid. As a solid, artificial diamond,
There are glass, silicon, graphite and various plastics. As an example, Fig. 1 shows DN on artificial diamond.
The structural formula in which A is immobilized is shown.

[0003]

Although the object of preventing the propagation of various bacteria can be achieved by storing in an environment of -30 ° C or lower, a lot of cost is required to maintain the storage environment. B) Storage in an environment of -30 ° C or lower requires a lot of space such as a storage device and storage container. C) It is difficult to organize when stored in an environment of -30 ° C or less. The present invention has been made to eliminate the above drawbacks. B) Preservation technology for cDNA-immobilized diamond chips 1) Preparation of cDNA-immobilized diamond chips. CDNA was immobilized on the diamond tip surface using RNA derived from rabbit β-globin. (Hereinafter cDN
A-immobilized chip) 2) A sample on which a cDNA-immobilized chip was immersed in sterilized water or ethanol was used as a sample. 3) Decompress the cDNA-immobilized chip in sterilized water or alcohol under the temperature shown in Table 1 (6 Pa = 6 ^* 10 ⁻⁶ N
/ Mm ² ) It was dried and used for storage. 4) After 120 days of storage, the activity was confirmed. The activity of the sample cDNA prepared in 3) was confirmed. a. The cDNA-immobilized chip was immersed in sterile water to prepare a sample. b. Using the above-mentioned cDNA-immobilized chip, a cDNA sample amplified by the PCR (enzymatic nucleic acid amplification) method was obtained.

[0004]

Means for Solving the Problems DNA is chemically bound onto a solid and it is dried rapidly. The present invention is a method for storing DNA having the above constitution.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. DNA is chemically bonded to artificial diamond, glass, silicon, graphite, and various plastics to be fixed (hereinafter, DNA-immobilized chip), and the DNA-immobilized chip is immersed in a liquid such as sterilized water or ethanol and rapidly dried. The present invention is a technique for treating and storing DNA with the above-mentioned constitution.

[0006]

EFFECTS OF THE INVENTION a) Since it can be stored in a dry state, it is possible to prevent the propagation of various bacteria. B) Storage device (low temperature refrigerator) by storing at room temperature
Is unnecessary. C) It can be stored in an environment such as a desk drawer (a cool and dark place), so it is easy to organize.

[Proof experiment of the invention] An experiment conducted for proving the present invention and its result. The temperature and pressure of the experiment were derived from the correlation diagram of water and ice (Fig. 2). c. The enzyme used is M-MLV (RNase H-). d. The primers used are 5'RACE and OGS70. e. PCR (enzymatic nucleic acid amplification) method 30 under the above conditions
Cycling was performed to prepare a sample for electrophoresis. The results are shown in FIG. Sample No. M was active in all samples except the spontaneous drying of No. 1 from the electrophoresis result (FIG. 2) of cDNA obtained by 100 bp ladder marker 5) PCR (enzymatic nucleic acid amplification) method. (B) Preservation technology for λDNA (double-stranded DNA prepared by lambda phage) -immobilized chip 1) A λDNA-immobilized chip is prepared by using a 21 kbp fragment (DNA fragment) obtained by treating λDNA with EcoR1 (restriction enzyme). Created. 2) Six λDNA-immobilized chips in sterilized water were freeze-dried at −2 ° C. (6 Pa = 6 ^* 10 ⁻⁶ N / mm ² ) and dried for storage. 3) Confirmation of activity after 120 days of storage a. The λDNA-immobilized chip was immersed in sterile water to prepare a sample. b. PC using a primer pair that amplifies 500 bp region
A sample amplified by the R (enzymatic nucleic acid amplification) method was obtained. c. PCR (enzymatic nucleic acid amplification) method 30 under the above conditions
Cycling was performed to prepare a sample for electrophoresis. The results are shown in FIG. Sample No. M is 100 bp ladder marker Sample No. 0 is the result by the conventional method 4) From the electrophoresis result of λDNA obtained by the PCR (enzymatic nucleic acid amplification) method (FIG. 3), the sample No. 0 by the conventional method and the present technology ( No significant difference is observed with Samples 1 to 6 according to the method for preparing stored samples). All samples were active, except for the number 1 natural dry.

Claims (3)

[Claims] 1. A technique in which a product obtained by chemically bonding DNA to a solid is rapidly dried by heating under reduced pressure and freeze-drying under reduced pressure and then provided for storage. 2. The reduced pressure heating and drying conditions according to claim 1 are 4
Decompression is 4 Pa (4^* 10^-6N / mm
^Two) It can be dried if: 3. The freeze-drying condition of claim 1 can determine the decompression limit according to the ice state diagram if it is 0 ° C. or lower.