WO2016195273A1

WO2016195273A1 - Anti-freezing composition

Info

Publication number: WO2016195273A1
Application number: PCT/KR2016/005082
Authority: WO
Inventors: 김재종; 임시규; 차선호; 원진상; 이현정
Original assignee: (주)제노텍
Priority date: 2015-06-03
Filing date: 2016-05-13
Publication date: 2016-12-08
Also published as: KR20160142530A; KR101845715B1

Abstract

The present invention relates to an anti-freezing composition which can stably preserve a nucleic acid, protein, etc. and, more specifically, to an anti-freezing composition which can simply and stably preserve a synthetic gene (oligonucleotide), genomic DNA, a PCR (RT/PCR) master mixture, a restriction enzyme, etc. by dissolving the synthetic gene (oligonucleotide), the genomic DNA, the PCR (RT/PCR) master mixture, the restriction enzyme, etc. in a solution comprising an anti-freezing agent which does not freeze even at a temperature of -20°C or below, thereby minimizing problems such as reduction in activity and inconvenience in use due to freezing and thawing processes having to be repeatedly performed whenever the synthetic gene (oligonucleotide), the genomic DNA, the PCR (RT/PCR) master mixture, the restriction enzyme, etc. are used.

Description

Cryoprotectant Composition

The present invention relates to a cryoprotectant composition for a biomaterial and a method of using the same, and more particularly, to a cryoprotectant composition capable of long-term storage of biomaterials such as nucleic acids and proteins without deactivation.

The present invention relates to a cryoprotectant capable of stably storing synthetic genes (oligonucleotides), genomic DNA, RNA, PCR (RT / PCR) master mixtures that are frequently used for PCR, cloning, hybridization, etc. Instead of distilled water or Tris buffer solution, which is a commonly used solvent, it is dissolved in a solution containing anti-freezing agents (AFAs) that do not freeze at temperatures below -20 ° C. The present invention relates to a new composition that can store biomaterials such as synthetic genes, genomic DNA, and PCR (RT / PCR) master mixtures simply and stably by minimizing the problems such as freezing / thawing process and reduced activity. .

In general, biomaterials such as proteins and DNA are poorly stored in aqueous solution, so they are stored at a cryogenic temperature below -20 ℃ rather than at room temperature to maintain activity. Will be kept. However, such repeated freezing and thawing alters the normal secondary structure of the protein, causing the protein to lose activity (Jameel F., Bogner R., Mauri F. and Kalonia D., J. Pharm Pharmacol, 49 (5). ), 472-477, 1997; Nardid O., Dyubko T. and Repina S., Cryobiology, 34 (2), 107-113, 1997). In the case of long-term storage of proteins, various methods of drying are used to stabilize them. Among these methods, some of the enzymes are frozen by lyophilization, in particular when enzymes such as restriction enzymes or DNA polymerase are stored. There is a problem that the activity is lost during the thawing process. In addition, depending on the type of material to be lyophilized, it must be completely frozen at various temperatures and rates. The freeze-drying apparatus required for this is expensive, and the freeze-drying process is relatively slow. This economic factor and the troublesome work make it difficult to use (see Korean Patent Registration No. 10-0857889).

Synthetic genes used for PCR are generally dissolved in TE buffer or distilled water, and stored at -20 ℃ or lower in consideration of long-term storage stability. Therefore, not only the inconvenience of dissolving each time use, but also repeated freezing and thawing cause a decrease in the activity of the synthetic gene, so it is necessary to pay close attention, such as the use in small amounts.

In the case of genomic DNA, for long-term storage, it is stably stored for 1-2 years at a temperature of 0-4 ° C. or less in an aqueous solution containing lyophilized or 70% ethanol. However, genomic DNA is also generally stored at 4 ° C in order to prevent a decrease in activity due to repeated freezing / thawing. In this case, however, it can be stably stored for up to 6 months, but if more than that, the purity of purified DNA Therefore, stability falls rapidly.

In the case of various enzymes, such as restriction enzymes, DNA polymerases, and reverse transcriptases, which are most frequently used in molecular biology experiments, a buffer solution for preservation in the state of 50% glycerol is generally used. The added glycerol lowers the freezing point of the solution to freeze enzyme To prevent this (see US Pat. No. 5,250,429). However, due to the high viscosity, the accuracy is poor when dispensing 1-2 μl.In the case of restriction enzymes, star activity occurs when the concentration of glycerol is used more than 5% of the total reaction solution. Therefore, there is a limit to the amount of restriction enzyme used. In addition, in order to minimize the cumbersome and experimental error of the preparation of the reaction solution, a master mixture containing a mixture of buffers, dNTPs, primers, and the like is often prepared and stored, but stable storage is limited.

Republic of Korea Patent Publication No. 2002-0013521 relates to a composition for preserving infectious recombinant adenovirus, containing adenovirus particles, characterized in that it comprises a buffer solution that is supplemented with glycerol and can maintain a medium pH of 8.0 to 9.6 Initiate a liquid or frozen composition.

Korean Patent Publication No. 2008-0087148 discloses a cryoprotective composition comprising a nanostructure, a liquid and at least one cryoprotectant comprising a nanometer sized core material surrounded by a regular fluid molecule of the liquid.

Republic of Korea Patent Publication No. 2005-0030416 relates to a cryopreservation method of clam floating larvae, the step of placing the clam floating larva into artificial seawater diluted with cryoprotectant to absorb the cryoprotectant, the clam absorbing the cryoprotectant A method of cryopreservation of clam floating larvae is provided by the step of placing the floating larvae at room temperature for an equilibrium time and cooling the clam floating larvae using a freezer after the equilibrium time.

The present invention relates to a composition and a method for cryopreservation at low temperature, and in addition to glycerol, the composition is different from the present invention because it uses a method of adding a buffer, a nanostructure, an artificial seawater, etc. to maintain the pH as an alkali. There is also a difference from the effect of the present invention, where freezing is prevented even at -30 ° C or lower.

Therefore, the present invention aims to provide a cryoprotectant composition which can stably store biomaterials such as nucleic acids or proteins even at low temperatures below freezing, and has no activity loss due to freezing / thawing.

In order to solve this problem, it is necessary to develop a composition capable of stably preserving the biomaterial through the identification of the type and composition ratio of the components included in the cryopreservation composition. Under this background, the present invention provides anti-freezing agents that do not freeze at temperatures below -20 ° C. instead of aqueous solutions in order to minimize problems such as the hassle of use and reduced activity that require repeated freezing and thawing every time they are used. , AFA) but also a component that does not inhibit the activity or stability of the biomaterial and the appropriate composition ratio between each component was confirmed and completed the present invention.

The cryoprotectant of the present invention

1) It is possible to prepare at concentration more than 2 times of working concentration,

2) Biomaterials such as oligonucleotide, genomic DNA, RNA, PCR (RT / PCR) master mixture and restriction enzymes within working concentration do not freeze even when stored at -20 ℃,

3) long-term storage at -20 ℃ does not affect the biological activity of the sample,

4) In the case of PCR reaction, even up to 30% of the reaction solution to be used (up to 6µl of 20µl reaction solution) does not affect the enzyme reaction.

5) Reduced star activity in restriction enzymes facilitates the use of higher amounts of restriction enzymes compared to 50% glycerol.

1 is a diagram showing the freezing point for each concentration of the cryoprotectant of the present invention.

Figure 2 is agarose gel photograph showing the effect of the cryoprotectant of the present invention on PCR using a pair of primers.

Figure 3 is agarose gel picture showing the maximum amount of the cryoprotectant of the present invention can be used for PCR.

4 is agarose gel photograph comparing the effect of the cryoprotectant of the present invention on the detection sensitivity of PCR.

5 is an agarose gel photograph comparing the stability of the deoxy oligonucleotide dissolved in the cryoprotectant of the present invention.

6 is an agarose gel photograph comparing the stability of genomic DNA dissolved in the cryoprotectant of the present invention.

Figure 7 is agarose gel photograph comparing the stability of the PCR master mixture containing the cryoprotectant of the present invention.

8 is an agarose gel photograph comparing the stability of the reverse transcriptase master mixture containing the cryoprotectant of the present invention.

9 is an agarose gel photograph comparing the freeze / thaw effect of the reverse transcriptase master mixture dissolved in distilled water.

10 is agarose gel photograph comparing the stability of the RNA dissolved in the cryoprotectant of the present invention.

11 is an agarose gel photograph comparing the freeze / thaw effect of RNA dissolved in distilled water.

12 is an agarose gel photograph comparing the stability of the restriction enzyme stored in the cryoprotectant of the present invention.

Figure 13 is agarose gel photograph comparing the star activity of the restriction enzyme stored in the cryoprotectant of the present invention.

14 is a result of measuring the stability and reactivity of the deoxyoligonucleotide and the PCR master mixture dissolved in the cryoprotectant of the present invention by real-time PCR.

Hereinafter, the structure of the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

실시예Example 1: 동결방지제 용액 제조 1: Preparation of cryoprotectant solution

Generally 50% glycerol solution is characterized by not freezing at -20 ℃. However, an aqueous solution containing 50% glycerol is not easily pippeted at 1-2 μl due to the viscosity of glycerol.

In general, a 50% glycerol solution has several drawbacks, since the laboratory must be able to prepare the storage solution at a concentration greater than or equal to the working concentration to dilute the sample to the desired concentration.

Therefore, in the present invention, in order to minimize the concentration of glycerol and increase the freezing point of the solution, at least one of ethanol or methanol was added, and a certain amount of ethanol and / or methanol was added to 20% and 10% of glycerol concentration to -20 ° C,- The cells were incubated at 30 ° C and -35 ° C for 24 hours. As a result, the freezing point of each solution was as shown in FIG.

As shown in FIG. 1, when ethanol 14% was added to 20% glycerol or 11% methanol was added to 20% glycerol, the solution did not freeze at -30 ° C., and 20% ethanol and 14% methanol were added to 10% glycerol. The solution did not freeze at 30 ° C. Therefore, by adding appropriate amounts of methanol and ethanol, the concentration of glycerol can be lowered to 10-20%, which is 24--40% ethanol or 20-40% glycerol at 20-40% glycerol, which is twice the working concentration. A new cryoprotectant solution can be prepared with a methanol concentration of 28%.

실시예Example 2: 동결방지제 용액이 2: cryoprotectant solution PCR에PCR 미치는 영향 Impact

To investigate the effect of 20-40% glycerol + 24-40% ethanol, or 20-40% glycerol + 22-28% methanol solution prepared in Example 1 on the reactivity of PCR (Polymerase Chain Reaction) As a result of investigating the reactivity of PCR by adding a certain amount of aqueous solution containing 10, 11, 12, 13% of methanol to glycerol and 13, 14, 15, and 16% of ethanol to 20% of glycerol for various types of primer pairs, As in AD of 2, it was confirmed that the PCR reaction was not affected at all. Figure 2 lane 1 is a DNA molecular weight marker, lane 2 is 20% glycerol 10% methanol, lane 3 20% glycerol methanol 11%, lane 4 20% glycerol methanol 12%, lane 5 20% glycerol 20 Methanol 13%, Lane 6 is 20% Glycerol 13%, Lane 7 is Glycerol 20% Ethanol 14%, Lane 8 is Glycerol 20% Ethanol 15%, Lane 9 is Glycerol 20% Ethanol 16%, Lane 10 Is distilled water. Primers used are 185s / 185as in FIG. 2, 5382s / 5382as in B in FIG. 2, 6174s / 6174as in C in FIG. 2, and D in HPRTs / HPRTas in FIG. Is shown in Table 1.

Human genomic DNA used for PCR was 200ng of DNA purified from peripheral blood by proteinase K / phenol-chloroform method. The composition of PCR reaction solution was prepared in 20µl reaction solution with 2µl of glycerol + methanol solution of each concentration or glycerol. 2 μl of ethanol solution and 10 pm Tris-HCl, pH 8.3, 40 mM KCl, 1.5 mM MgCl ₂ , 50 mM / mL BSA, 250 pm each of dNTP, sense primer, antisense primer 20 pmoles were used respectively.

실시예Example 3: 3: PCR에PCR 사용할 수 있는 동결방지제 용액 최대량 Maximum amount of cryoprotectant solution available

20 μl sample for optimal concentration of 20% glycerol + 12% methanol (abbreviated as "AFA 1") and 20% glycerol + 15% ethanol (abbreviated "AFA 2") As a result of investigating the maximum amount that can be used for the PCR reaction, it was found that up to 5 µl of both "AFA 1" and "AFA 2" had no effect on PCR (FIG. 3). 3A is "AFA 1", FIG. 3B is "AFA 2", lane 1 is a DNA molecular weight marker, lane 2 is added 1 µl, lane 3 is added 2 µl, lane 4 is added 3 µl, lane 5 is 4 µl Addition, lane 6 is 5 µl, lane 7 is 6 µl, lane 8 is 7 µl, lane 9 is 8 µl, lane 10 is 9 µl, lane 11 is distilled water and lane 12 is a negative control. Primers used for PCR are A1s / H4as and the nucleotide sequence and PCR conditions are shown in Table 1.

실시예Example 4: 동결방지제 용액이 4: cryoprotectant solution PCR의PCR 검출 민감도에 미치는 영향 Impact on detection sensitivity

In order to investigate the effects of "AFA 1" and "AFA 2" on the detection sensitivity of PCR, compared to distilled water (control) for DNA diluted to a certain concentration, it did not appear to have a significant effect as shown in FIG. Figure 4A is "AFA 1", Figure 4B is "AFA 2", Figure 4C is distilled water (control), lane 1 is a DNA size marker, lane 2 is TB (tuberculosis) DNA, lane 3 is TB DNA 1ng, lane 4 is 100 pg of TB DNA, 10 pg of DNA is

lane

5, 1 pg of DNA is

lane

6, 7 is TB DNA is

lane

7, 10 is TB DNA is 10fg lanes, 9 is TB DNA 1fg is

lane

9, 10 is TB DNA is 100ag lane 11 Is a negative control.

Primers used for PCR are TBs / TBas and the sequencing and PCR conditions are listed in Table 1.

실시예Example 5: 동결방지제에 보관된 5: stored in cryoprotectant 올리고뉴클레오타이드의Of oligonucleotides 안정성 stability

To investigate the stability at -20 ° C of oligonucleotides stored in "AFA 1" and "AFA 2", a pair of primers were dissolved in "AFA 1", "AFA 2" and distilled water (control), respectively, -20 ° C. After 30 days, 60 days, 90 days and 120 days, respectively, the PCR was carried out to examine the PCR reactivity, and all three primer pairs did not affect the reactivity (FIG. 5). In Figure 5

lanes

1, 2, 3, 4 and 5 are in distilled water (control),

lanes

6, 7, 8, 9 and 10 are in "AFA 1" and

lanes

11, 12, 13, 14 and 15 are in "AFA". 2 ",

lanes

1, 6 and 11 are 0 months,

lanes

2, 7 and 12 are 1 month,

lanes

3, 8 and 13 are 2 months,

lanes

4, 9 and 14 are 3 months,

lanes

5, 10 and 15 Is stored at -20 ℃ for 4 months.

Primers used for PCR is 185s / 185as and the nucleotide sequence and PCR conditions are listed in Table 1.

실시예Example 6: 동결방지제에 보관된 유전체 DNA의 안정성 6: Stability of Genomic DNA Stored in Cryoprotectants

In order to investigate the stability of genomic DNA dissolved in "AFA 1" and stored at -20 ° C, the result of the comparative experiment with genomic DNA dissolved in distilled water and stored at 4 ° C was dissolved in "AFA 1" as shown in FIG. The stored genomic DNA had no problem with activity after 6 months.

Lanes

1, 2, and 3 of FIG. 6 are stored in distilled water,

lanes

4, 5, and 6 are stored in distilled water, and 2 µl of "AFA 1" is added during PCR, and

lanes

7, 8, and 9 are "AFA 1". "It was kept in.

Primers used for PCR are A1s / H4as and the nucleotide sequence and PCR conditions are shown in Table 1.

실시예Example 7: 동결방지제에 보관된 7: stored in cryoprotectant PCRPCR 마스터 혼합물의 안정성 Stability of the master mixture

To compare the stability of the PCR master mixtures (10X buffer, 10 mM dNTP, 5U Taq DNA polymerase), the PCR master mixtures were dissolved in distilled water and stored at 4 ° C and "AFA 1" and stored at -20 ° C for 3 weeks. The reactivity of was compared. As a result of diluting human genomic DNA into 1/10, 1/20, 1/50, and 1/100, the detection sensitivity was examined. As shown in FIG. In Figure 7,

lanes

1, 2, 3 and 4 are dissolved in "AFA 1" and stored at -20 ° C,

lanes

5, 6, 7 and 8 are dissolved in distilled water, then stored at -35 ° C,

lanes

9, 10, 11 and 12 are dissolved in distilled water and stored at 4 ° C. for 1 week,

lanes

13, 14, 15 and 16 are dissolved in distilled water and stored at 4 ° C. for 2 weeks,

lanes

17, 18, 19 and 20 are dissolved in distilled water and 4 ° C. For 3 weeks,

lanes

1, 5, 9, 13, and 17 diluted 1/10 of human genomic DNA,

lanes

2, 6, 10, 14, and 18 diluted human genomic DNA to 1/20 Figures 1,

lanes

3, 7, 11, 15, and 19 are 1/50 dilution of human genomic DNA,

lanes

4, 8, 12, 16, and 20 are 1/100 dilution of human genomic DNA.

Primers used for PCR are ACTs / ACTas and the sequencing and PCR conditions are listed in Table 1.

실시예Example 8: 동결방지제에 보관된 8: Stored in cryoprotectant 역전사효소Reverse transcriptase 마스터 혼합물의 안정성 Stability of the master mixture

To compare the stability of the reverse transcriptase master mixture (10X buffer, 10 mM dNTP, 400U RTase, 100U RNasin), the reactivity of the reverse transcriptase master mixture dissolved in distilled water and stored at 4 ° C and -20 ° C and 20% glycerol and 12% methanol Cryoprotectant 1 containing (AFA 1), reverse transcriptase master mixture stored in cryoprotectant 2 (hereinafter AFA 2) containing 20% glycerol and 15% ethanol was compared. As a result, as shown in Figure 8, the distilled water 4 ℃ storage activity was significantly reduced after 1 week, distilled water -20 ℃ and "AFA 1" -20 ℃ storage was not reduced until 3 weeks. In Figure 8

lanes

1, 2, 3, and 4 are dissolved in distilled water and stored at -20 ℃,

lanes

5, 6, 7 and 8 are dissolved in distilled water and stored at 4 ℃,

lanes

9, 10, 11 and 12 were dissolved in "AFA 1" and stored at -20 ° C,

lanes

1, 5 and 9 for 0 weeks,

lanes

2, 6 and 10 for 1 week,

lanes

3, 7 and 11 for 2 weeks,

lanes

4, 8 and 12 were stored for 3 weeks.

When dissolved in distilled water and stored at -20 ℃, the freeze / thaw process should be performed every time it is used.In this case, the reverse transcriptase master mixture dissolved in distilled water and stored at -20 ℃ to compare the decrease in activity is 5, 10, After 20, 30 freeze / thaw cycles, activity was examined. As a result, it was confirmed that activity is completely eliminated from one time as shown in FIG. 9. In Figure 9

lanes

1 and 2 are dissolved in "AFA 1" and stored at -20 ℃,

lanes

3 and 4 dissolved in distilled water and then freeze / thaw 5 times,

lanes

5 and 6 freeze / thaw 10 times,

lanes

7 and 8 are 20 freeze / thaw cycles and

lanes

9 and 10 are 30 freeze / thaw cycles.

In the case of reverse transcriptase master mixture, it can be seen that it can be used stably without loss of activity when dissolved in "AFA 1" and stored at -20 ° C.

The RNA used for the RT / PCR reaction was HCV RNA purified from the serum of the patient, and the primers used for PCR were HCV RT / 1st sense primer, HCV RT / 1st antisense primer, HCV 2nd sense primer, and HCV 2nd antisense primer. Sequence and RT / PCR conditions are listed in Table 2.

실시예Example 9: 동결방지제에 보관된 RNA의 안정성 9: Stability of RNA Stored in Cryoprotectants

As a result of comparing the stability of the RNA dissolved in "AFA 1" and stored in -20 ℃ and the reactivity of the reverse transcriptase master mixture stored in -20 ℃ dissolved in distilled water, as shown in Figure 10A -20 ℃ and "AFA 1" At -20 ° C, there was no decrease in activity until 3 weeks.

Lanes

1 and 2 of Figure 10A are stored in "AFA 1",

lanes

3 and 4 are stored in distilled water, lane 5 is a negative control. However, after 11 months of comparison, as stored in distilled water and stored at -20 ℃ as shown in Figure 10B completely reduced the activity, stored in -20 ℃ after dissolved in "AFA 1" activity compared to the newly extracted RNA It was confirmed that there is no effect at all. In FIG. 10B, lane 1 is freshly extracted control RNA,

lanes

2 and 3 are stored in distilled water at −20 ° C.,

lanes

4 and 5 are stored in “AFA 1,” and

lanes

3 and 5 are 1/2 of

lanes

2 and 4. RNA is diluted.

In addition, when dissolved in distilled water and stored at -20 ℃, each use must go through the freeze / thaw process.In this case, the RNA dissolved in distilled water and stored at -20 ℃ to compare the decrease in

activity

1, 5, 10 and After 15 freeze / thaw processes, the activity was examined. As shown in FIG. 11, the activity was completely eliminated from the first time. In FIG. 11,

lanes

1 and 2 are stored in “AFA 1”,

lanes

3 and 4 are stored in distilled water, and then frozen / thawed once,

lanes

5 and 6 are frozen / thawed 5 times, and

lanes

7 and 8 are frozen / thawed. Ten defrost cycles,

lanes

9 and 10 were repeated 15 freeze / thaw cycles.

HCV RNA purified from the serum of the patient was used for the RT PCR reaction. The primers used for PCR were HCV RT / 1st s, HCV RT / 1st as, HCV 2nd s and HCV 2nd as, and the sequence and RT / PCR conditions are shown in Table 2. It is described in.

실시예Example 10: 동결방지제에 보관된 제한효소의 안정성 10: Stability of restriction enzyme stored in cryoprotectant

To compare the stability of restriction enzymes stored in cryoprotectants, five restriction enzymes of Ava I, BamH I, EcoR I, Hind III, and Sal I were added to 50% glycerol, "AFA 1", and "AFA 2" solutions, respectively. After storage at −20 ° C., irradiation was conducted over 0, 2, and 6 weeks. Each restriction enzyme was used as 4U for Ava I, 8U for BamH I, 6U for EcoR I and Hind III, and 8U for Sal I. The amount of each solution contained in 10 μl reaction solution was 1 μl. After reacting at 37 ° C. for one hour, 8% agarose gel was confirmed. As a result of six weeks of irradiation, as shown in FIGS. 12A, 12B, and 12C, all of 50% glycerol, "AFA 1", and "AFA 2" showed no activity at all. In Figure 12

lanes

1, 6 and 11 are Ava I,

lanes

2, 7 and 12 are BamH I,

lanes

3, 8 and 13 are EcoR I,

lanes

4, 9 and 14 are Hind III,

lanes

5, 10 and 15 Sal I,

lanes

1, 2, 3, 4 and 5 are 50% glycerol,

lanes

6, 7, 8, 9 and 10 are “AFA 1” and

lanes

11, 12, 13, 14 and 15 are “AFA 2” to be.

Lambda DNA was used for the restriction enzyme reaction, and 150 ng was used for the 10 µl reaction solution.

실시예Example 11: 동결방지제에 보관된 제한효소의 스타 활성 11: Star activity of restriction enzyme stored in cryoprotectant

EcoR I was used to compare star activity in 50% glycerol and "AFA 1", "AFA 2" solutions. 1 μl, 2 μl, and 3 μl of EcoR I contained in each solution were added to the 10 μl reaction solution, reacted at 37 ° C. for 30 minutes, and then confirmed on an 8% agarose gel. As a result, 50% glycerol and "AFA 2" showed star activity from 2 μl, and 3 μl of "AFA 1" also showed star activity, but for "AFA 1", 50% glycerol and "AFA 2" as shown in FIG. It can be seen that star activity is significantly reduced compared to. In Figure 13

lanes

1, 2 and 3 are 50% glycerol,

lanes

4, 5 and 6 are "AFA 1",

lanes

7, 8 and 9 are "AFA 2" and

lanes

1, 4 and 7 are added 1 μl, lanes 2

microliters

2, 5, and 8 were added, and 3 microliters of

lanes

3, 6, and 9 were added.

The DNA used for the restriction enzyme reaction was lambda DNA, and 100 ng was used for the 10 µl reaction solution.

실시예Example 12: 동결방지제 용액이 실시간 12: cryoprotectant solution in real time PCRPCR (Real-time (Real-time PCRPCR )에 미치는 효과)

In order to investigate whether the cryoprotectant solution used in the stability test in Examples 5 and 7 also affects the reactivity and stability of real-time PCR, oligonucleotides and fluorescent pigment-labeled probes and PCR master mixtures were added to "AFA 1". After dissolution, the test was started and stored for 6 months and 9 months at -20 ° C, respectively, and then real-time PCR reactivity was confirmed.

Real-time PCR was performed with multiplex PCR on two targets, in addition to two pairs of primers, two probes labeled with FAM and Cy5 were used. Sequences and PCR conditions are shown in Table 3.

DNA used for PCR is each recombinant plasmid DNA containing a portion of the nucleotide sequence of the HLA-B27 gene and β-actin gene, and the plasmid DNA isolated from the E. coli culture using the separation purification kit is quantified by UV spectrophotometer. The molecular weight was calculated and converted into copy number. Real-time PCR reactivity was measured by Bio-Rad (USA) CFX96 Touch Real-Time PCR Detection System, the composition of the PCR reaction solution 10X oligonucleotide mixture dissolved in "AFA 1" (HLA-B27 s of 5uM concentration each) And 2 μl of HLA-B27 as, HLA-B27 p at 2.5 uM concentration, ICs at concentrations of 3.5 uM and IC as IC concentration at 1 uM concentration, and 5X PCR master mixture (5X PCR) dissolved in "AFA 1" 4 μL buffer, 5 mM dNTP mixture, 5 U Taq DNA polymerase), template DNA was positive control mixture dissolved in TE buffer (pH 8.0) (HLA-B27 at 50,000copies / μl and β-actin at 5,000copies / μl) 2 μl of recombinant plasmid) solution and 12 μl of distilled water were added to adjust the final volume to 20 μl.

14A shows the start of the test, B is stored at -20 ° C for 6 months, and C is stored at -20 ° C for 9 months.

The present invention freezes biomaterials comprising at least one of protein or nucleic acid, including a combination of at least one of ethanol or methanol and glycerol, at a temperature of 0 ° C. or lower and −40 ° C. or higher, without storing the activity without freezing. It relates to an inhibitor composition.

In the present invention, the cryoprotectant composition is a combination of 10-40% (v / v) ethanol or 10-30% (v / v) one or more kinds of methanol and 10-40% (v / v) glycerol It relates to a cryoprotectant composition comprising a.

In addition, the present invention relates to a cryoprotectant composition, characterized in that the biomaterial is at least one member selected from the group consisting of polymerase, restriction enzyme, reverse transcriptase, DNA, RNA, oligonucleotide and dNTP.

In addition, the present invention is a combination of the cryoprotectant composition of at least one of 20 to 40% (v / v) ethanol or 20 to 30% (v / v) methanol and 20-40% (v / v) of glycerol It includes, and relates to a cryoprotectant composition that can be stably stored at a temperature of 0 ° C -40 ° C or higher even when diluted to 1/2 during operation.

In addition, the present invention includes a combination of at least one of 20-40% (v / v) ethanol or 20-30% (v / v) methanol with 20-40% (v / v) glycerol, Even when diluting to 1/2 during operation, the cryoprotectant composition that can be stably stored at a temperature of 0 ° C. or lower than −40 ° C. or more; and one or more biomaterials of protein and nucleic acid; And to a method of use.

The cryoprotectant composition of the present invention can stably store biomaterials such as nucleic acids or proteins even at low temperatures below zero, and can be used in fields such as medicine, biotechnology, and forensics because there is no loss of activity due to freezing / thawing.

Claims

A cryoprotectant composition which can store a biomaterial comprising at least one of protein or nucleic acid containing a combination of at least one of ethanol or methanol and glycerol at a temperature of 0 ° C. or lower and −40 ° C. or higher without losing activity.
The method according to claim 1,

The cryoprotectant composition comprises a cryoprotectant comprising a combination of 10-40% (v / v) ethanol or 10-30% (v / v) one or more of methanol and 10-40% (v / v) glycerol. Composition.
The method according to claim 1,

The biomaterial is a cryoprotectant composition, characterized in that at least one selected from the group consisting of polymerase, restriction enzyme, reverse transcriptase, DNA, RNA, oligonucleotide and dNTP.
The method according to claim 1,

The cryoprotectant composition comprises a combination of at least one of 20-40% (v / v) ethanol or 20-30% (v / v) methanol and 20-40% (v / v) glycerol, Freeze-protective composition that can be stably stored at a temperature of 0 ° C. or lower and −40 ° C. or higher even when diluting to 1/2 of an hour.
It contains a combination of at least one of 20-40% (v / v) ethanol or 20-30% (v / v) methanol and 20-40% (v / v) glycerol, with 1/2 of the work. Cryopreservative composition that can be stably stored at a temperature of 0 ° C -40 ° C or higher even when diluted; and

One or more biomaterials of proteins and nucleic acids; mixing and storing in a volume ratio of 1: 1.