KR20170103436A - Massive gene purification kit for prompt molecular diagnosis - Google Patents

Abstract

Most of the conventional diagnostic kits require 5 hours or 12 hours to diagnose diseases, so it takes a long time to isolate the gene and only a small number of genes can be separated. Accordingly, rapid and convenient massive gene separation technology is needed. When a specific solvent and salt are added to a gene, the gene-salt complex becomes large and heavy. Therefore, it is possible to easily isolate genes by filtering with a syringe filter or a vacuum filter.

Description

[0002] Massive gene purification kit for prompt molecular diagnosis < RTI ID = 0.0 >

TECHNICAL FIELD The present invention relates to a gene purification kit for mass and rapid molecular diagnosis, and a kit for mass purification and ultrapure water gene purification for research.

The kit for gene purification for laboratory research mainly uses an affinity purification method and has a limitation in the amount of purified water. It is estimated that the kit for gene purification for laboratory research is estimated to be about $ 1 billion worldwide, and about 80% is owned by Qiagen, and the domestic market is estimated to be about 60 billion won. Although the domestic gene products are developed in the research gene purification kit, they are still dominated by foreign products and depend on imports for about 80%.

The conventional gene purification kit is made of a resin type or an adsorption filter type, and it is necessary to purify a limited amount of genes according to the capacity of the resin, the type and capacity of the adsorption filter, and a large number of genes have to be obtained by repeated experiments when necessary.

It is an object of the present invention to provide a gene purification kit capable of solving the problems of the prior art and capable of purifying genes easily and quickly.

In order to successfully develop a kit for rapidly isolating genes, the present inventor has searched for existing diagnostic kits, and in order to diagnose diseases using most conventional kits, a short time of 5 hours, a long time of 12 hours is required, And that the experiment should proceed. Accordingly, the present invention aims to develop a kit capable of purifying genes necessary for molecular biology experiments and the like within 30 minutes in a large amount.

In molecular biology experiments and molecular diagnostics using PCR, gene purification is an essential experiment, and the reagents and kits needed here are largely dependent on imports. Current methods of gene purification use a silica membrane or resin. The adsorption filter method is widely used for small gene purification, and it is a method that uses the property that the (-) charge of the gene binds to the (+) charge of silica when the salt concentration is high in a specific environment. The adsorption filter method has advantages in that it can rapidly purify the gene and is generally used because it can obtain a pure gene. Most gene purification products developed and marketed in Korea utilize this method. A method of gene purification using a resin is a method of purifying a gene by binding a (+) charge molecule capable of adsorbing a gene to the surface of a solid bead, and it can purify the gene to the purest one , And Qiagen's products account for 90% of the total market. It is used for purification of genes used for experiments requiring high purity such as mass gene purification or transfection.

In addition, pretreatment of the sample is essential for gene purification. The pretreatment of the sample exposes the gene in the cell by treating the sample for a long time at a high temperature using a reagent such as Protease K. These exposed genes are purified by adsorption filters or resin columns in specific environments, such as high salt concentrations or specific pH ranges.

In order to solve the problems of the prior art,

A) disrupting cells in a specimen sample and extracting a gene;

(B) adding alcohol having 1 to 3 carbon atoms and a chaotropic salt to the gene extracted in step (a), stirring the solution, and passing the solution through a filter;

C) washing the filter after step b); And

And d) eluting the gene attached to the filter after the c) washing step.

In addition, the present invention relates to a mass gene purification method for rapid molecular diagnosis characterized in that the alcohol in step b) is at least one selected from the group consisting of methanol, ethanol, propanol and isopropanol.

The present invention also relates to a method for producing a chaotropic salt according to the present invention, wherein the chaotropic salt of step b) is sodium salt including sodium acetate (final concentration 0.1-0.5M), sodium chloride (final concentration 0.05-0.5M) M), ammonium acetate (final concentration: 1 to 2M), and the like.

Further, the present invention is characterized in that the filtration membrane of the filter in the step b) is one selected from the group consisting of Teflon, MCE (Mxed Cellulose Ester), PES (Polyethersulfone), nylon, nitrocellulose and PVDF (Polyvinylidene Fluoride) To a method for mass gene purification.

Also, the present invention relates to a method for mass gene purification for rapid molecular diagnosis, wherein the Teflon filter filtration membrane in step b) is one selected from PTFE-J, PTFE-H, PTFE-T and PTFE-D.

In addition, the present invention relates to a method for mass gene purification for rapid molecular diagnosis, wherein the gap of the filter membrane in step b) is 0.1 to 5.0 탆. If the pore size is less than 0.1 탆, the amount of the sample that can pass through is reduced to 3 ml or less. If the pore size exceeds 5.0 탆, the DNA molecules can not be passed through the filter and the DNA can not be recovered.

The present invention also relates to a mass gene purification kit for rapid molecular diagnosis comprising a buffer for cell disruption, an alcohol having 1 to 3 carbon atoms, a chaotropic salt, a filter for gene filtration, a washing buffer and an elution buffer.

In addition, the present invention relates to a mass gene purification kit for rapid molecular diagnosis, wherein the alcohol having 1 to 3 carbon atoms is at least one selected from the group consisting of methanol, ethanol, propanol and isopropanol.

In addition, the present invention relates to a mass gene purification kit for rapid molecular diagnosis characterized in that the chaotropic salt is at least one selected from a sodium salt, lithium chloride and ammonium acetate.

In addition, the present invention is characterized in that the filtration membrane of the filter is one selected from the group consisting of Teflon, MCE (Mxed Cellulose Ester), PES (Polyethersulfone), nylon, nitrocellulose and PVDF (Polyvinylidene Fluoride) The present invention relates to a tablet kit.

The present invention also relates to a kit for mass gene purification for rapid molecular diagnosis, wherein the Teflon filtration membrane of the filter is selected from among PTFE-J, PTFE-H, PTFE-T and PTFE-D.

In addition, the present invention relates to a large-scale gene purification kit for rapid molecular diagnosis, characterized in that the filtration membrane pore of the filter is 0.1 to 5.0 탆.

The gene purification kit of the present invention can be utilized for mass purification of a gene used in a kit for gene purification for laboratory research or gene therapy and overcomes the limitation of purification amount which is the limit of the adsorption filter or resin type gene kit You can purify as many genes as you want.

In addition, the kit of the present invention permits early diagnosis of disease occurrence by allowing the gene to be purified within 10 minutes to one hour in the field so that an experiment for diagnosis can be performed.

In addition, when the kit of the present invention is used for the purification of a gene for research, a large amount of genes can be obtained in a short time as compared with gene purification using an existing product.

Further, when the kit of the present invention is used, more than 90% of the genes can be purified.

In addition, by using the kit of the present invention, trace genes in the sample can be purified.

1 is a conceptual diagram showing the principle of the present invention.
FIG. 2A is a result of a first experiment to search for the degree of DNA binding and recovery according to the material and pore size of a filter in the method of the present invention.
Lane 1: 1kb marker
Lanes 2 and 7: PTFE-H, 0.22 탆
Lanes 3 and 8: PTFE-D, 0.22 탆
Lanes 4 and 9: PVDF, 0.22 탆
Lane 5, 10: MCE, 0.22 탆
Lanes 6 and 11: PTFE-J, 0.22 탆
Lane 12: Qiagen Midi
FIG. 2B is a graphical representation of the results of FIG. 2A.
FIG. 3A is a result of a second experiment to search for the degree of DNA binding and recovery according to the filter material and pore size (0.45 μm) in the method of the present invention.
Lane 1: 1kb marker
Lanes 2, 6, 10: NYLON, 0.45 mu m
Lanes 3, 7 and 11: PTFE-H 0.45 탆
Lanes 4, 8, 12: MCE 0.45 탆
Lanes 5, 9 and 13: PTFE-D 0.45 탆
Lane 14: Qiagen Midi
Figure 3B is a graphical representation of the results of Figure 3A.
4A is an experimental result for establishing an optimal method of extracting plasmid DNA using a filter.
Lane 1: 1kb marker
Lane 2: EtOH, washed once, eluted once
Lane 3: EtOH, washed once, eluted twice
Lane 4: EtOH, washed once, eluted three times
Lane 5: EtOH, washed twice, eluted once
Lane 6: EtOH, washed twice, eluted twice
Lane 7: EtOH, washed twice, eluted three times
Lane 8: IPA, one wash, one elution
Lane 9: IPA, 1 wash, 2 elution
Lane 10: IPA, washing once, elution three times
Lane 11: IPA, twice washing, one elution
Lane 12: IPA, two washings, two elution
Lane 13: IPA, washing twice, elution three times
FIG. 4B is a graph showing the result of FIG. 4A.
5 is an experimental result for checking whether the method of extracting plasmid DNA using a syringe filter is applicable to various plasmids.
Lane 1: pET32b E1
Lane 2: pET32b E2
Lane 3: pET22b E1
Lane 4: pET22b E2
Lane 5: pET23d E1
Lane 6: pET23d E2
Lane 7: pEGFP E1
Lane 8: pEGFP E2.
FIG. 6 is a graph showing an experiment for obtaining plasmid DNA according to the culture amount of E. coli containing plasmid DNA.
Fig. 7 is a graph showing the effect of the P < RTI ID = 0.0 > C < / RTI > SF plasmid & The yield and purity of various plasmid DNAs are shown. DNA yield and purity were determined by measuring the absorbance at 260 nm and 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific).
E1: first elution, E2: second elution.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the drawings and specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.

1 is a conceptual diagram showing the principle of the present invention. When ethanol and salt are added to the sample containing the gene, crystals are formed, and the gene binds to the salt to increase the size of the complex. When the filter is passed through a filter having micro-pores, the liquid phase passes through the filter. However, It is possible to isolate and purify the gene by a simple process.

FIG. 2A shows the results of the first experiment for exploring the degree of DNA binding and recovery according to the material and pore size of the syringe filter membrane in the method of the present invention. The cultured Escherichia coli cells were inoculated with buffers 1, 2 and 3, disrupted and centrifuged. Ethanol and salts were added to the resulting supernatant, which was then filtered using five kinds of syringe filters. After filtering the sieved filter, the sieved filter was washed with washing buffer, eluted with elution buffer, and the DNA was recovered and electrophoresed (lanes 7-11).

Lane 1: 1 kb marker, lanes 2 and 7: PTFE-H, 0.22 탆,

Lanes 3 and 8: PTFE-D, 0.22 탆, lanes 4 and 9: PVDF, 0.22 탆,

Lane 5, 10: MCE, 0.22 탆, lane 6, 11: PTFE-J, 0.22 탆,

Lane 12: Qiagen Midi.

FIG. 2B is a graphical representation of the results of FIG. 2A.

As a result of lanes 2-6 in Fig. 2a, it was found that plasmid DNA binds to all of the five kinds of syringe filters. However, in the results of lanes 7-11, it was confirmed that the recovery rate of the plasmid DNA varies depending on the material of the syringe filter. In other words, depending on the type of syringe filter, DNA binding is good and recovery is good (lanes 3 and 8), while DNA binding is good but DNA recovery is not done at all (lanes 5 and 10). Thus, it was confirmed that the type of the syringe filter greatly influences the experimental results.

FIG. 3A shows a result of a second experiment to search for the degree of DNA binding and recovery according to the syringe filter material and pore size (0.45 μm) in the method of the present invention. The cells were disrupted by adding Buffer 1, 2, and 3 to the cultured Escherichia coli cells, and the cells were centrifuged. Ethanol and salts were added to the obtained supernatant, and the mixture was passed through five kinds of syringe filters. Samples were obtained by washing and twice eluting the after filter and syringe filter through the syringe filter (the first elution was lane 6-9 and the second elution was lane 10-13).

Lane 1: 1kb marker

Lanes 2, 6, 10: NYLON, 0.45 mu m

Lanes 3, 7 and 11: PTFE-H 0.45 탆

Lanes 4, 8, 12: MCE 0.45 탆

Lanes 5, 9 and 13: PTFE-D 0.45 탆

Lane 14: Qiagen Midi

Figure 3B is a graphical representation of the results of Figure 3A.

3A and 3B, the degree of DNA binding and the recovery rate were different depending on the material of the syringe filter. In other words, PTFE-D filter showed the best results when compared with other filters having different material and pore size.

4A is an experimental result for establishing an optimal method of extracting plasmid DNA using a syringe filter.

Lane 1: 1kb marker

Lane 2: EtOH (ethanol), washing once, elution once

Lane 3: EtOH, washed once, eluted twice

Lane 4: EtOH, washed once, eluted three times

Lane 5: EtOH, washed twice, eluted once

Lane 6: EtOH, washed twice, eluted twice

Lane 7: EtOH, washed twice, eluted three times

Lane 8: IPA (isopropyl alcohol), washing once, elution once

Lane 9: IPA, 1 wash, 2 elution

Lane 10: IPA, washing once, elution three times

Lane 11: IPA, twice washing, one elution

Lane 12: IPA, two washings, two elution

Lane 13: IPA, washing twice, elution three times

FIG. 4B is a graph showing the result of FIG. 4A.

Cells were disrupted by adding buffer solutions 1, 2, and 3, and extracted with ethanol (lane 2-7) or IPA (isopropanol) (lane 8-13) as a solvent for precipitating DNA from the cell lysate (Lane 2-4, 8-10), and washing twice (lanes 5-7, 11-13) after binding the sample solution containing DNA to the syringe filter . DNA was eluted from the syringe filter three times and recovered. The results were compared with each other. Overall, DNA yields were uniform, but 70 IPA was the best in terms of purity.

5 is an experimental result for checking whether the method of extracting plasmid DNA using a syringe filter is applicable to various plasmids. In the case of a high copy plasmid in which the method of the present invention for extracting plasmid DNA using a syringe filter is applicable even in the case of a low copy plasmid (lanes 1-6) 7-8).

Lane 1: pET32b E1, lane 2: pET32b E2,

Lane 3: pET22b E1, lane 4: pET22b E2,

Lane 5: pET23d E1, lane 6: pET23d E2,

Lane 7: pEGFP E1, lane 8: pEGFP E2.

Among the description of each lane, E1 means the first elution, and E2 means the second elution.

FIG. 6 is a graph showing the plasmid DNA uptake according to the amount of cultured cells in three replicate experiments of E. coli containing plasmid DNA having each characteristic. In the case of pEGFP, pET 23d plasmid DNA showing high replication number, 16 After culturing and extracting time, 252 ug and 150 ug of plasmid DNA were obtained, respectively, and when converted into ml, it corresponds to 28 ug and 16.6 ug. In the case of pET 22b and pET 32b corresponding to low copy number plasmids, 230 μg and 200 μg were obtained in a 50 ml culture medium, which corresponds to 4.6 μg and 4 μg, respectively, in terms of ml.

Fig. 7 is a graph showing the results obtained by using 10 < RTI ID = 0.0 > ml < / RTI > O / N cultures using a P & C SF plasmid- The yield and purity of various plasmid DNAs are shown. DNA yield and purity were determined by measuring the absorbance at 260 nm and 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). In the case of pETEP32b, pET22b, pET23b with high copy number and pET23b with pETFP (1.73 and 1.74, respectively), 1.8dp recognized as pure DNA were extracted. In the case of pEGEP, the first extract (E1) and two (E2) were extracted close to 1.8, and no difference was found between the low copy number and the high copy number plasmid.

E1: first elution, E2: second elution.

Claims (15)

A) disrupting cells in a specimen sample and extracting a gene;
(B) adding alcohol having 1 to 3 carbon atoms and a chaotropic salt to the gene extracted in step (a), stirring the solution, and passing the solution through a filter;
C) washing the filter after step b); And
And d) eluting the gene attached to the filter after the c) washing step.
The method according to claim 1,
Wherein the filter is a syringe filter equipped with a vacuum filter or a filtration membrane.
The method according to claim 1,
Wherein the alcohol in step (b) is one or more of methanol, ethanol, propanol and isopropanol.
The method according to claim 1,
Wherein the chaotropic salt in step b) is at least one selected from the group consisting of sodium salt, lithium chloride and ammonium acetate.
The method according to claim 1,
Wherein the filtration membrane of the filter in the step b) is one selected from the group consisting of Teflon, MCE (Mxed Cellulose Ester), PES (Polyethersulfone), nylon, nitrocellulose and PVDF (Polyvinylidene Fluoride) Way.
The method of claim 5,
Wherein the Teflon filtration membrane of the filter of step b) is one selected from PTFE-J, PTFE-H, PTFE-T and PTFE-D.
The method according to claim 1,
Wherein the filtration membrane of the filter of the step b) has a pore size of 0.1 to 5.0 탆.
Mass genetic purification kit for rapid molecular diagnosis, including alcohols of 1 to 3 carbon atoms, chaotropic salts and filters for gene filtration.
The method of claim 8,
Wherein the alcohol having 1 to 3 carbon atoms is at least one of methanol, ethanol, propanol, and isopropanol.
The method of claim 8,
Wherein the chaotropic salt is at least one selected from sodium salt, lithium chloride and ammonium acetate.
The method of claim 8,
Wherein the filtration membrane of the filter is one selected from the group consisting of Teflon, MCE, PES, nylon, nitrocellulose, and PVDF.
The method of claim 11,
Wherein the Teflon filtration membrane of the filter is one selected from PTFE-J, PTFE-H, PTFE-T and PTFE-D.
The method of claim 8,
Wherein the filtration membrane of the filter has a pore size of 0.1 to 5.0 占 퐉.
The method of claim 8,
Wherein the gene filtering filter is a syringe filter equipped with a vacuum filter or a filtration membrane.
The method of claim 8,
A buffer for cell disruption, a washing buffer, and an elution buffer.

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