Patent Application
20070254294
The present invention relates to a method for storing DNA, a method for analyzing the DNA stored by the aforementioned method and products using the methods. More particularly, the invention relates to a method for storing DNA in a stabilized state at room temperature for an extended time, to a method for analyzing the DNA stored by the aforementioned method and to a DNA ID card such as a DNA card made of paper or a DNA card made of plastic, which are produced using the aforementioned storing and analyzing methods.
The sum of all genes carried by organisms is referred to as genomes. A human genome is reported to be composed of 3 billion bases and have about 30,000 genes. Recently, the human genome project for decoding base sequences of the full human genome has been completed, thereby allowing epoch-making improvement in diagnosis and treatment of intractable diseases by using genes. So to speak, the time of personalized medicine and predictive medicine has begun.
Life phenomena are determined by three subjects: (1) genetic information of genomic DNA, (2) gene transcription and (3) expressed proteins. In order to understand and analyze life phenomena, and to develop methods for diagnosis and treatment of diseases, it is important to precisely analyze the aforementioned three subjects. The sum of genes in an individual is referred to as genes or genomes, and accordingly the sum of transcribed genes (i.e. mDNA) and the sum of expressed proteins in an individual is referred to as transcriptome and proteome, respectively. Recently, studies on automated analysis of such information in total are progressed actively. For automation of analysis, a microarray or a biochip in particular is helpful, and the representative examples thereof include a DNA chip and a protein chip.
As for a general method for qualitative and quantitative analysis of genetic information of genomic DNA, hybridization analysis, sequencing analysis, DNA microarray or chip, or the like via PCR, PCR-RFLP, cloning and library production, southern blotting or the like (Sambrook, J. & Russell, D. W., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (2001)) may be mentioned.
When a person's DNA is tested, it is possible to understand the person's health and give a diagnosis of whether the person is contracted to various diseases such as cancer or infection, as well as to predict possibilities of henceforth outbreaks of genetic diseases and transmittance to the person's child. Further, DNA test is the most accurate way to prove one's identity and kinship such as paternity. It is also helpful in proving genealogical lines or family trees. For that reason, DNA test has been used inevitably as a means for the forensic medicine and for discriminating suitability when donating the bone marrow, other internal organs or the like. Further, by analyzing single nucleotide polymorphism (SNP), it may help in predicting possibility of henceforth outbreaks of diseases, reaction to drugs or the like.
As the necessity of storing DNA for future DNA test was recognized, each country has readily established DNA banks, and the number of samples in the DNA bank tends to increase largely in the reason of storing DNA relative to travelers insurances or accident insurances, storing DNA of the U.S. armed forces or the like. However, in storing personal DNA at a national organization or a commercial organization, there may have a problem such as misuse of personal genetic information. Moreover, in order to store DNA safely for an extended time by means of the existing technology, there are need for freezers and specific reagents, thus the cost for storing DNA is large, accordingly.
In fact, when DNA separated and purified from a cell is left to stand at room temperature, there may be a problem of the DNA being rapidly degraded or segmented by deoxyribonuclease (DNAse). Therefore, a method for storing DNA for studies in gene is important. Currently, as for the method of storing or carrying DNA, which is used widely, a method of storing DNA as a liquid state by purifying DNA separated from body fluid such as blood, cells or tissues; a method of storing in a freezer; a method of freeze-dry storing; a method of storing in 2-propyl alcohol; and the like may be mentioned. However, there are many disadvantages in these methods in the viewpoint of costs and losses. The method of storing the purified DNA as a liquid state is at ease for using the DNA immediately, but there is a problem that the DNA is easily damaged when stored for an extended time. The method of storing in a freezer and the method of freeze-drying has a problem that the DNA may be damaged from repeated melting and freezing in the process of using the sample. Further, the method of storing using a reagent such as alcohol is also troublesome in the process of pre-treatment and post-treatment as well as other methods. Moreover, the most important problem in the above processes is that DNA must be purified first from the body fluid such as blood for storage.
The above-described methods for storing DNA require special expensive equipments such as ultra-low temperature freezer, liquid nitrogen, and the like. Thus, it is disadvantageous in that the methods may impose burden in the economical point of view, the methods require processes of pre-treatment and post-treatment when reusing the stored DNA for analysis, and the method could damage DNA from repeated processes of freezing and defrosting.
In addition to the aforementioned methods, recently, several commercialized products such as a FTA card or a card manufacture by Whatman plc., and the like for the purpose of storing plasmid DNA at room temperature are introduced. However, this method has limitations to its effectiveness, and it is suggest to store the DNA at −15° C. or at −20° C. instead of room temperature itself, thus there is no big advantage over the original methods, but rather is disadvantageous in the increase of cost matter.
Therefore, it is an important object in the field of molecular genetics and medicine to develop a possible method for protecting DNA from decomposing by deoxyribonuclease via forming a stable bind with DNA, storing DNA in a stabilized state at room temperature, and further applying directly to the studies and analyses using DNA. Ideal methods of storing should include: (1) a method of storing stably at room temperature without having the DNA degraded; (2) a method should be simple; (3) a method should be cost effective; (4) DNA storing should not only be for the separated and purified DNA itself, but also be for samples containing body fluid such as blood and various DNAs without having damages on the DNA; (5) various DNA samples should be stored in a limited space; (6) an individual should possibly store one's personal DNA; and (7) there should not have any problems in the henceforth analysis on various genes, and these genetic tests should be helpful to actual diagnosis or studies. However, methods or products that satisfy these conditions have not been introduced so far, and reports on the products for storing personal DNA together with the persons genetic information could not be found.
Chitosan is a biologically produced polymer which is excellent in stability, biodegradability and suitability on a living body, and is used widely in the field of medicine. When chitosan is treated with an acid, it forms a water-soluble chitosan salt which becomes charged with a strong positive charge, thus in recent years, the chitosan salt has been noted as a prominent delivering means of drugs, proteins and DNAs. In particular, water-soluble chitosan forms a strong bind with DNA, which is an anionic substance, for the protection of the DNA. Therefore, the water-soluble chitosan may serve as a medium substance for delivering genes.
Chitosan is a copolymer of glucosamine and N-acetylglucosamine and obtained from chitin. Chitin is abundant in crustacean such as crabs or shrimps, and is a substance of natural polymers which is most abundant next to cellulose. Chitosan is a generic term of cationic polymers which is obtained by subjecting chitin to deacetylation with a strong base (Errington N., Harding S. E., Varum K. M., Illum L., Int. J. Biol. Macromol., 15, 1123-1127 (1993)).
Chitosan is not dissolved in neutral or basic pH, while forms chitosan salts or cationic chitosan when reacted with acids such as glutamic acid, hydrochloric acid, lactic acid or the like. The chitosan salt is dissolved in water, and its solubility is proportionate to the degree of deacetylation and pH. In the case of water-soluble chitosan, it becomes positively charged by adding a proton to an amine group. Further, water-soluble chitosan can be made into a freeze-dried state.
Water-soluble chitosan has properties of forming a film or gel with a strong positive charge. In this regard, chitosan is used in purifiers for filthy water, heavy metals and potable water, and antifungal substances. It is also used as raw materials of drinks, health foods and cosmetics. In addition, chitosan is used as an important means in the field of pharmacy. For example, chitosan is used in the wide ranges for delivering of vaccine, delivering of DNA, facilitation of delivering of proteins, peptides and drugs via production by purification and a controlled drug release system, a gel, a film, a wetting agent, a coating agent, a microsphere, a microcapsule, a bioadhesives and a mucous membrane, and the like (Illum L., Pharmaceutical Research, 15, 1326-1331 (1998)).
Chitosan is very safe and show no side effects caused by immunization. When chitosan is absorbed into a living body, it is degraded to N-acetylglucosamine with lysozyme, after which is used in the synthesis of glycoproteins, and then the residual resultant is discharged as a form of carbon dioxide (Chandy T., Sharma C. P., Biomat. Art. Cells Art. Org., 18, 1-24 (1990)).
The most important property of water-soluble chitosan or cationic chitosan according to the invention is that it forms a strong bind with DNA, which is an anionic substance, such that it can be used as a protection and a delivering means of DNA, namely, as a gene delivering substance. In the experiment of using a plasmid DNA, it is reported that chitosan functions as to stably preserve DNA without decomposing by DNA nucleases such as DNase I and DNase II, and that when a complex of chitosan and plasmid DNA is injected into in vitro cultured cell, it triggers expression of a delivering gene (Lee M., Nah J-W., Kwon Y., Koh J. J., Jo K. S., Kim S. W., Pharmaceutical Research, 18(4), 427-531 (2001)). Gene delivering effect is determined by (1) the molecular weight of chitosan and (2) the ratio of chitosan to DNA, in particular, the nitrogen/phosphate ratio of the phosphate molecule of anionic DNA to the nitrogen molecule of cationic chitosan (Borchard G., Advanced Drug Delivering Reviews, 52, 145-150 (2001)).
It is reported that when a plasmid DNA is left to stand at room temperature as it is, the plasmid DNA is decomposed in several hours; however, when a plasmid DNA is kept in a form of a complex with chitosan, it is possible to store the plasmid DNA for 3 months or more (Leong K. W., Mao H. Q., Turong-Lee V. L., Roy K., Walsh S. M., August J. T., J. controlled Rel., 53, 183-193 (1998)). Meanwhile, there has been a report in that when a complex of chitosan and plasmid DNA is stored in a freeze-dried state, the gene delivering effect can be continued until after 4 weeks (Mao H. Q., Turong-Lee V. L., August J. T., Leong K. W., Proc. Intl. Symp. Control. Rel. Bioact. Mater., 24, 671-772 (1997)).
However, the studies on possibility of stably storing large genomic DNA of a mammal with chitosan or the studies on possibility of stably storing DNA with chitosan for an extended time at room temperature has not been reported hitherto. Moreover, products produced to store DNA using chitosan have not been manufactured. Also, possibility of performing an analysis of gene expression using chitosan-bound DNA and subject on storing DNA in a mixed form of chitosan and body fluid, other than DNA itself, such as blood has not been reported. Further, products for storing, carrying and analyzing genomic DNA of a human or an animal using chitosan, other than the products of the invention, have not been released.
[Technical Problem]
An aspect of the invention is that it provides a method for storing and carrying DNA sample in a stabilized state at room temperature using water-soluble chitosan.
Another aspect of the invention is that it provides a complex of water-soluble chitosan and stabilized DNA which can be used in various methods of DNA test.
A further aspect of the invention according to the method for storing DNA using chitosan of the invention is that it provides a chitosan solution with an appropriate property and concentration that can form a stable bind with DNA in order to possibly carry and store DNA for an extended time at room temperature, easily study and analyze DNA and easily be subject to application.
A still further aspect of the invention is that it provides a method for preparation of a paper type card containing chitosan (a DNA card) that can store combined chitosan and DNA as a liquid state and a number of DNA samples in a minimum space, a method for performing a genetic analysis such as PCR, PCR-RFLP, cloning, sequencing analysis and southern blotting, microarray test and the like, and a method for separating chitosan and DNA from their conbined state. In this case, DNA card can be largely classified into Type 1 and Type 2: Type 1 stores DNA itself on a paper type card containing chitosan, and Type 2 stores bio-sample containing DNA such as blood other than DNA itself on a paper type card comprising chitosan and a cell lysis buffer. In both cases, the object is to store DNA in stabilized state for an extended time at room temperature and to perform various genetic analyses thereafter, if necessary.
Another aspect of the invention is that it provides a DNA identification card (DNA ID card) in which DNA sample of an individual is stored in a form of mixture with chitosan, and genetic information of an individual obtained by performing tests such as a genetic test for personal identification, a HLA genotyping test or the like is saved on a magnetic bar or a chip on the plastic card. The plastic DNA ID card can be used for storing personal information such as various credit cards and cash cards, entrance cards to important organizations, bio-passport, military forces or the like, or sharing genetic information for an organ transplant such as bone-marrow transplantation.
[Technical Solution]
In the invention, a new method for storing large genomic DNA of animals and plants was studied, and as a result, it was confirmed that when water-soluble chitosan is mixed with DNA, a stable bind is formed so as to protect DNA from decomposing by deoxyribonuclease and can be stored for an extended time in a stabilized state at room temperature, and thus completed the invention.
The conditions to be achieved by the methods and products of the invention are as follows:
1) DNA should be stored in a stabilized state without degrading at room temperature;
2) DNA samples herein should include genomic DNA and cDNA of humans, animals, plants and bacteria, and plasmid DNA;
3) the method should be simple and should not require special equipments or instruments to be used;
4) the cost should be economical;
5) DNA storing should not only be for the separated and purified DNA itself, but also be for bio-samples containing body fluid such as blood and various DNAs without having damages on the DNA;
6) DNA sample should be stored using a simple medium such as a paper or a card;
7) a number of DNA samples should be stored in a limited space;
8) an individual should possibly store one's personal DNA;
9) with respect to the DNA samples, which was stored and transported by the aforementioned methods and products, various genetic analyses such as PCR, PCR-RFLP, hybridization, sequencing analysis, SNP analysis and the like should be performed accurately and simply even after an extended time, and should be helpful to studies as well as various clinical examinations; and
10) the DNA ID card should function as DNA bank for storing personal DNA, and also store genetic information of the person together therewith.
Hereinafter, the invention will be described in detail in reference to the drawings.
In the invention, in order to form a stable complex with DNA, to protect DNA from decomposing by deoxyribonuclease, and to store DNA in a stabilized state at room temperature, a water-soluble chitosan solution with an appropriate property and concentration, a paper type card or a PCR kit containing such solution and a plastic type DNA ID card and the like are used.
It is preferable that chitosan used in the invention has a molecular weight of 10 to 500 kDa. Chitosan having the molecular weight of less than 10 kDa has problems in forming a stable bind with DNA, and chitosan having the molecular weight exceeding 500 kDa may interfere with analyzing gene expression using DNA. Further, chitosan with the degree of deacetylation of 60% or more is preferred, and when the degree of deacetylation is less than 60%, a problem in water-solubility of chitosan occurs, thus it is difficult to form a stable bind with DNA. The concentration of a prepared aqueous solution of chitosan, in terms of weight to volume ratio (w/v), is 0.02% to 1%, and preferably 0.1%. The mixture ratio at the time of mixing an aqueous solution of chitosan and an aqueous solution of DNA, in terms of the weight ratio of chitosan in the aqueous solution of chitosan to DNA in the aqueous solution of DNA, is 1:0.5 or more, and preferably 1:0.5 to 1:3 (Examples 1 and 2).
DNA/chitosan bound product according to the invention is stored at a temperature from room temperature to −70° C., which is wide in its ranges, and is stored in a dark place without moisture.
Meanwhile, DNA stored in a form of binding with water-soluble chitosan according to the invention is stably preserved even with degradation by deoxyribonuclease is occurred, thus can be used for genetic analysis (see Example 2).
According to the invention, DNAs which can be stored in a mixture with chitosan include genomic DNAs of humans, animals or plants, bacteria, and plasmid DNA, and preferably include cDNA. The size of DNA which can be stored in a mixture with chitosan varies from several tens to several billions of bases.
Meanwhile, a liquid mixture of DNA bound with water-soluble chitosan according to the invention is preserved stably, and the effectiveness of a method for storing DNA of the invention was easily confirmed by performing tests such as polymerase chain reaction (PCR), cloning, blotting, sequencing analysis and the like.
According to the invention, a card for DNA storage (Type 1 DNA card) can be prepared by mixing an appropriate concentration of chitosan and an uric acid (particularly, the volume ratio of the mixture of 0.1% to 1% (w/v) of the water-soluble chitosan solution to the concentration of 0.5 mM to 20 mM of the uric acid is 1:1), and adsorbing the mixture on a paper for blotting, and then drying. Using the aforementioned card, a number of DNA can be stored for extended time and carried in a minimum space at room temperature. As for the paper used in the preparation of the card for storing DNA, various papers for chromatography or papers for blotting that can be used commercially may be used, and the thickness of the paper is preferably about 0.3 mm to 1.2 mm. The storage temperature of the card for storing DNA is suitably room temperature, but lowering the temperature to −20° C. and −70° C. is allowable, and storing for 6 months or more is possible at room temperature (Example 5).
When performing PCR in the invention, in the case where a mixture of DNA and chitosan is a liquid, the mixture itself is used as a template, and in the case where it is in a card form, a fragment of the card is cut and used as a temperate to perform a general method of PCR amplification, cloning and sequencing analysis. Consequently, it is confirmed that the DNA was preserved stably enough to perform the method of genetic analysis using the stored DNA according to the method of the invention (Example 5).
According to the invention, a card for storing DNA (Type 2 DNA card) can be prepared by adsorbing a mixture of an appropriate chitosan solution with a cell lysis buffer comprising Tris, EDTA, SDS and uric acid (particularly, Tris (8 mM), EDTA (0.5 mM), SDS (0.1% w/v) and uric acid (2 mM)) on a paper for blotting, and then drying. Using the aforementioned card, DNA can be stored for an extended time and carried at room temperature as a bio-sample state containing DNA such as blood (Example 6).
Meanwhile, DNA stored using the DNA card of the invention is preserved stably, and then tests such as PCR, PCR-RFLP, cloning, sequencing analysis and the like can be performed henceforth (Examples 7 to 10).
Further, in order to perform southern blotting, the complex of DNA and chitosan should be separated so that DNA may move on the agarose gel. Thus, DNA is separated by using a sulfate-based cationic salt that has no damage on DNA and has no effect on the results of southern blotting. Examples of the aforementioned sulfate-based cationic salt include sodium dodecyl sulfate (SDS), sodium octyl sulfate (SOS) or cetyltrimethyl-ammonium bromide (CTAB) (Example 9).
As an application of the DNA storage method using chitosan in the invention, a PCR kit was prepared wherein Taq polymerase, a primer, dNTP and buffer and the like were added to one tube with chitosan (all in one tube), and a sample such as DNA or serum was added to the tube to lead to PCR reaction. Characteristically, this PCR kit can be used for both of DNA storage and PCR assay (Example 11).
In addition, the invention provides a DNA ID card in which DNA sample of an individual is stored in a form of mixture with chitosan, and genetic information of an individual obtained by performing a genetic test for personal identification or a HLA genotyping test is saved on a magnetic bar on the plastic card (Examples 12 and 13). The plastic DNA ID card can be used as a personal DNA bank as well as a personal genetic information bank for personal identification, personal organ transplant, personal clinical diagnosis or the like.
The present invention will be further illustrated with examples below. However, the scope of the present invention is not limited thereto.
To establish suitable properties, concentration and mixing condition of chitosan for forming a stable complex when mixed with DNA, water-soluble chitosan of various concentrations was mixed with whole DNA isolated from monocyte of normal adult blood in various ratios, and then mobility shift assay of DNA bound to chitosan was carried out through agarose gel electrophoresis. Furthermore, mobility shift assay of DNA bound to chitosan was carried out in the same manner as for genomic DNA of mouse hepatic tissue and plasmid DNA (pCMV-beta-galactosidase) of 4.0 kb size. The method for experiment and the results are as follows.
A. DNA Isolation and Preparation
Whole DNA of the cell from normal adult leukocyte was isolated, the experiment was performed using tertiary distilled water according to a known method (Sambrook J & Russell D W, Molecular cloning: a laboratory manual, Cold Spring Harbor Press. 2001:7.1-7.88).
The peripheral venous blood was collected in a tube containing sodium citrate (vaccutainer CTAD tube, catalogue No #367946, Becton & Dickinson, USA), and the plasma and buffy coat layer of the monocyte were separated through centrifuge. The monocyte DNA, most of which is lymphocyte, was isolated using a QIAamp DNA blood minikit (Qiagen, Germany) in the following method.
20 μl of protein kinase K (QIAGEN protease) was put into 1.5 ml-microcentrifuge tube, 200 μl of the buffy coat layer and 200 μl of buffer AL were sequentially added thereto, and mixed by vortex for 15 seconds. It was left to stand at 56° C. for 10 minutes and centrifuged to collect the solution stuck to the tube cap. 200 μl of ethanol was added thereto, mixed by vortex for 15 seconds, and all of the solution was added to a QIAamp spin column, and was centrifuged at 8,000 rpm for one minute. Then, a 2 ml-collecting tube was inserted into the column, 500 μl of a buffer AW1 was added thereto, and centrifuged again at 8,000 rpm for one minute. Thus-obtained filtrate was discarded, and a new tube was provided, and further centrifuged at a maximum speed for one minute. Again, a new 1.5-ml microcentrifuge tube was inserted, 200 μl of distilled water or a buffer AE was added thereto, left to stand at room temperature for one minute, and then further centrifuged at 8,000 rpm for one minute to elute DNA. Thus-isolated DNA was measured on the concentration using a spectrophotometer, and the A260/A280 ratios were compared in order to find the purity of the isolated DNA. At this time, the DNA purity should be such that A260/280 is 1.6 to 1.8 upon spectrophotometer measurement. From the above method, 6 μg on average of pure DNA can be obtained from 200 μl of human blood.
B. Preparation of Chitosan
If a water-soluble chitosan is a product of which the deacetylation degree is 60% or more and the molecular weight is 10 kDa to 500 kDa, it can be used for the applications of the present invention, specifically for forming a stable complex with DNA. Many water-soluble chitosan products corresponding to these are commercially available. Among them, in the present invention, a water-soluble chitosan product of which the average deacetylation rate is 90.1% and the average molecular weight is about 300 kDa, was purchased from Jakwang Co. Ltd. (Ansungsi, Republic of Korea), and dissolved in sterilized tertiary distilled water. Besides this chitosan, other water-soluble chitosan having similar properties may be also used.
C. Method For Preparation of a Complex of Chitosan and DNA
The water-soluble chitosan was dissolved in sterilized tertiary distilled water, and prepared at various concentrations of from 0.02% to 0.05%, 0.1%, 0.25%, 0.5% and 1% of weight versus volume ratio (w/v) for test. Here, DNA was diluted with sterilized tertiary distilled water to the concentrations of 500 nμg/μl and 1 μg/μl, and then used.
In a 1.5-ml centrifuge tube, a DNA solution and the chitosan solution of various concentrations as described above were mixed at various volume ratios, and left to stand at room temperature for 15 minutes to induce formation of a complex.
D. Mobility Shift Assay of a Complex of DNA and Chitosan Through Agarose Gel Electrophoresis
The DNA/chitosan complex obtained by mixing a 1 μg/μl concentration of genomic DNA isolated from normal adult monocyte and plasmid DNA, and the water-soluble chitosan solutions of various concentrations at various ratios were loaded on 0.8% agarose gel according to the above method, and subjected to electrophoresis at 100 V. The results were shown in
To ascertain if the water-soluble chitosan can protect DNA from deoxyribonuclease (DNAse), and establish the suitable conditions, DNA which was treated with a chitosan solution, and which was DNA not treated, were treated with deoxyribonuclease, respectively, and the effect was observed through agarose gel electrophoresis.
The method and results were as follows. The same experiment was performed repeatedly with genomic DNA of human lymphocyte and genomic DNA of mouse hepatic tissue.
Eight tubes were divided into two groups. In one group, chitosan (20 g) dissolved in distilled water and DNA (10 g) were mixed in a microcentrifuge tube (1.5 ml) to produce a complex. In the other group, only DNA (10 g) was added to the tube without chitosan. To each of the tubes of the first group and the second group, 5 μl of deoxyribonuclease (DNAse I), which was dissolved at a concentration of 1 unit/l , was added, and then distilled water was added to 100 μl pf a final volume. Then, they were held for reaction in 37° C. incubator for 0 minute, 20 minutes, 40 minutes and 60 minutes, respectively. Immediately after the reaction finished, 25 μl of the stop solution was added and well mixed to inactivate DNAse I. To each of the four tubes wherein the reaction was inactivated, 110 μl of TE buffer (10 mM Tris, 0.1 mM EDTA) was added and mixed, and then they were held for reaction at 60° C. overnight. Then, 150 μl of phenol/chloroform was added and mixed by vortex for one minute, and centrifuged at room temperature for 5 to 8 minutes. Then, a supernatant was taken and put into a new microcentrifuge tube, and extracted. 300 μl of anhydrous EtOH and 15 μl of 3M NH40AC were added thereto, and left to stand previously at −70° C. in a deep freezer for 20 to 30 minutes. Then, the resultant was centrifuged at 4° C. in 12,000 rpm for 20 minutes, and the liquid was discarded by suction. The settled pellet was washed again with 70% ethanol, and dried, and dissolved in 20 μl of distilled water, which was free from DNAse. 10 μl (about 1 μg DNA) of the resultant was taken from thus-obtained total eight tubes, respectively, and subjected to electrophoresis on 1% agarose gel. The results were shown in
From
Using the liquid complex of the water-soluble chitosan and DNA prepared according to Examples 1 and 2, PCR was performed to ascertain if the target gene is appropriately amplified, and thereby to ascertain if the DNA storage method of the present invention has no harmful effect on the gene assay, and to establish suitable conditions for using DNA stored by the DNA storage method according to the present invention in PCR. For human leukocytic genomic DNA, PCR was performed with the target of each ? actin gene. The method and results were as follows.
PCR reaction was performed using 100 ng of the subject DNA as a template by adding 10 pmol of the forward primer and the reverse primer of the target gene, respectively, and further adding 2.5 mM deoxynucleotide (dNTPs), 1 unit Taq polymerase and 10× polymerase buffer (500 mM KCl, 100 mM Tris-Cl, 15 mM MgCl2, 0.1% gelatin). PCR was performed using GeneAmp 2700 thermal cycler (Perkin-Elmer Biosystems, Inc., USA), firstly under the condition of first 95° C. for 5 minutes, then forty times under the condition of 95° C. for 30 seconds, 55C for 30 seconds and 72C for 30 seconds, and finally at 72° C. for 7 minutes, to give PCR products.
With each template of genomic DNA samples of human and mouse prepared by mixing 0.1% chitosan and DNA (1 μg/μl) at a volume ratio of 1:1, and DNA which was not mixed with chitosan, PCR was performed for beta-Actin gene. The results of electrophoresis on 1.5% agarose gel were shown in
From the above results, it was found that the water-soluble chitosan is nearly completely bound to DNA, and preserves DNA stably from the attack of deoxyribonuclease. In the present example, it was ascertained if when the complex of the water-soluble chitosan and DNA was stored in the liquid form at room temperature for a long time, the stored DNA is kept with no cleavage to make it possible to perform gene amplification and assay. It was performed by PCR assay as follows.
1 μl of 0.1% the water-soluble chitosan solution (w/v) and 1 μl of genomic DNA of monocyte of normal adult blood (500 nμg/μl) were mixed to form a complex, and 8 μl of distilled water was further added to adjust the final volume to 10 μl. Thus, 24 samples were prepared in the centrifuge tubes and stored at room temperature. 1 μl of the same leukocyte DNA, which is not treated with chitosan, was adjusted to a final volume 10 μl to prepare 9 samples. Three of them were stored at −70° C. and the remaining six samples were stored at room temperature. For the DNAs which were mixed with the water-soluble chitosan and stored in the tube at room temperature, PCR reaction was performed according to the method of Example 3 for three DNA samples, respectively at the time point of one week, two weeks, three weeks, four weeks, three months, six months, nine months and one year, respectively to ascertain if the target gene is appropriately amplified. For the three samples which were stored at −70° C. among the DNA samples which were not mixed with chitosan, PCR was performed after three months, and for each three DNA samples which were stored at room temperature, PCR was performed after three months and one year, respectively.
From
Such results showed that the method of storing DNA in the liquid as bound to the water-soluble chitosan according to the method of the present invention, can keep DNA stably for at least one year, that the stored DNA has no problem in being used in PCR assay, and that the method can store DNA stably similarly to the conventional storage method of the deep freezer. The storage temperature in the present example varies from room temperature to −70° C. It is preferably stored in non-humid dark place.
To store and carry the multiple sample DNAs at room temperature in a minimum space, and help with automatic assay, a card wherein chitosan is absorbed on a suitable paper has been developed, which was designated as a DNA card. The DNA card is in various kinds, and among them, a paper card of which the use is storing DNA itself, was designated as Type 1 DNA card.
To select the most effective concentration of the water-soluble chitosan solution and appropriate thickness of the paper, the following experiment was performed.
Various kinds of papers for blotting were submerged in 0.02%, 0.1% or 0.25% (w/v) concentration of the water-soluble chitosan solution during a sufficient time period, and dried to be used in producing DNA cards. At this time, the paper is selected from papers for blotting which are commercially available from various companies such as Whatman plc. (UK) or Schleicher & Schuell GmbH (Germany), and has a suitable thickness. In the present invention, papers for blotting of 0.3 mm, 0.9 mm and 1.2 mm in thickness were purchased from Whatman plc. and used. The kind and size of the DNA card for DNA storage with chitosan can be prepared variously depending on use. The blotting paper on which chitosan is adsorbed, was cut to the same size of 24-well, 96-well and 384-well plate (multiwell-plate) (8.1 cm 12.3 cm), which are generally well used, and the well and partition were printed and marked on the paper like the plate. On each of the wells, a DNA sample was dropped with a pipet, and stored for one week to one year or more at room temperature. Thus, one DNA card can store 24 to 96, to 384 pieces of DNA samples.
A method of producing the water-soluble chitosan-submerged DNA card is as follows.
A 0.3 mm thick paper for blotting from Whatman plc. was sterilized and dried in an autoclave at high temperature. Then, 0.1% (w/v) concentration of water-soluble chitosan solution and 2 mM uric acid solution, which were previously produced, were mixed at a volume ratio of 1:1, and the paper was submerged during a sufficient time period, and then dried to produce Type 1 DNA card.
On the Type 1 DNA card prepared as above, human genomic DNA in which human genome DNA was spotted and stored for a long time, was dropped, and stored for a certain period. From the stored DNA card, a card fragment of about 1 mm to 2 mm in diameter was taken using a punch, a forceps or a pincette, and added to a tube for performing PCR. With a template of this card fragment, PCR for beta-Actin gene was performed according to the method of Example 3. The method of this PCR reaction was as follow.
1. From a chitosan-submerged paper card wherein DNA is stored, a fragment of about 1.2 mm in diameter was taken using a punch, etc. and added to a tube for PCR.
2. 200 μl of a TE buffer (10 mM Tris-Cl, 0.1 mM EDTA, pH=8.0) was added, mixed, and then left at room temperature for 5 minutes. Thereafter, the TE buffer was completely removed using a pipet.
3. The second process was repeated twice.
4. It was dried at room temperature for 1 hour or at 56° C. for 10 minutes.
5. To the card fragment which was treated by the above processes, a PCR sample of a suitable composition was added and beta-Actin gene was PCR amplified according to the method of Example 3.
This Type 2 DNA card was produced by adsorbing chitosan and a cell lysis buffer on various kinds of papers for blotting and drying it. This Type 2 DNA card was designed to be used to store samples as cell-containing body liquid such as blood, and then amplify them by PCR for gene assay. Onto this Type 2 DNA card, human blood was dropped, and after a certain period, the performance was compared and analyzed by PCR.
The paper card for blotting prepared as in Example 5 was submerged in the water-soluble chitosan solution and cell lysis buffer during a sufficient time period and dried to produce a DNA card. At this time, the papers for blotting of 0.3 mm, 0.9 mm and 1.2 mm in thickness were purchased from Whatman plc. and used. A method of producing Type 2 DNA card for storage as blood is as follows.
0.3 mm thick paper for blotting from Whatman plc. was sterilized at high temperature and dried. Then, 0.1% (w/v) concentration of the water-soluble chitosan solution and cell lysis buffer (0.5 mM EDTA, 8 mM Tris-Cl, 2 mM uric acid, 1% (w/v) SDS), which were previously produced, were mixed at a volume ratio of 1:1, and the paper was submerged during a sufficient time period, and then dried to produce Type 2 DNA card.
From the above DNA card, a card fragment of about 1 mm to 2 mm in diameter was taken, and added to a tube for performing PCR as in Example 5. With a template of this card fragment, PCR for beta-Actin gene was performed. The method of this PCR reaction was as follows.
1. From a chitosan-submerged paper card wherein DNA is stored, a fragment of about 1.2 mm in diameter was taken using a punch, etc. and was added to a tube for PCR.
2. 200 μl of a washing solution (GG purification reagent; 0.5 mM EDTA, 8 mM Tris-Cl, 2 mM Uric acid, 1% (w/v) SDS) was added, mixed and left at room temperature for 5 minutes. Then, the washing solution was completely removed using a pipet.
3. The second process was repeated three times.
4. Then, 200 μl of a TE buffer (10 mM Tris-Cl, 0.1 mM EDTA, pH=8.0) was added, mixed and left at room temperature for 5 minutes. Then, the TE buffer was completely removed using a pipet.
5. The fourth process was repeated twice or three times.
6. It was dried at room temperature for 1 hour or at 56° C. for 10 minutes. 7. To the card fragment which was treated by the above processes, a PCR sample of a suitable composition was added, and beta-Actin gene was PCR amplified in the same manner as in Example 2.
As results of the experiment, it was found that for all of Type 2 DNA cards, the target gene was strongly expressed, and that the thicker the paper was such as 0.9 mm or 1.2 mm, the more PCR products were produced. In addition, when stored as adsorbed on the DNA card of the invention, beta-Actin gene was strongly amplified in PCR even after a long time such as three months to one year at room temperature. Such results show that DNA sample can be stably preserved at room temperature for a long time as blood with no need to isolate and purify DNA when stored in the DNA card according to the invention, and can be assayed after a long time. Therefore, it was found that the DNA card of the invention is useful for storing and carrying simply DNA samples as blood at room temperature for a long time, and further also useful for various gene assays such as PCR or the like.
PCR was performed for a chitosan/DNA complex which was stored as a liquid at room temperature for a long time according to Example 4, and the obtained product was cloned. Then, it was ascertained by automatic base sequencing if the DNA stored according to the method of the invention can be stably kept and used after the storage.
With a template of a genomic DNA sample of an adult colic tissue, which was stored as mixed with 0.1% the water-soluble chitosan according to the method of the invention at room temperature for three months, PCR was performed for adenomatous polyposis coli (APC) gene. The product was cloned in a plasmid vector, and then searched again by base sequencing. The method is as follows.
It is important to adjust the PCR product of gene to a suitable concentration in order to be used as a template in the sequencing reaction. In the invention, 10 ng APC gene was used. To a PCR tube, the PCR product of each exon of APC gene, 3.2 pmol of either one of a forward or reverse primer and 8 μl of the reaction mixture (Terminator ready reaction mix; Perkin-Elmer, USA) were added, and sterilized distilled water was added to a 20 μl final volume and well mixed. Cycle sequencing reaction was performed for the mixture using a GeneAmp 2700 thermal cycler 25 times of 96° C. for 10 seconds, 50° C. for 5 seconds and 60° C. for 6 minutes. The obtained reaction product was precipitated with ethanol and centrifuged to remove fluorescence labeled dideoxynucleotide (ddNTPs) in the free primer and the reaction mixture (terminator ready reaction mix), and dried. The thus-obtained DNA was mixed with a mixture of formamide 25 mM EDTA (pH 8.0) : blue dextran and 10 μl of a loading buffer, and denatured in boiling water for 5 minutes. Then, the sample was placed on the ice, and the denatured DNA sample was added to each well of plates, which had been cast previously with 5.5% long ranger gel (BMA, catalogue No., USA). Electrophoresis was performed for 2 to 4 hours and base sequencing was conducted using a software of an ABI Prism 377 automatic sequencer (Perkin-Elmer Biosystems, USA).
The results of the automatic base sequencing for APC gene are shown in
As results of the sequencing, it was found that normal APC gene was exactly amplified, and therefore the liquid complex of DNA and chitosan according to the method of the invention preserve DNA so stably even after storage at room temperature for a long time to conduct cloning and base sequencing, which means that it is also useful for testing gene mutation and searching cancer.
It was ascertained if for the DNA sample stored as adsorbed on the DNA card at room temperature for a long time, cloning for the product obtained by PCR and automatic base sequencing can be appropriately conducted.
With a template of a genomic DNA sample of a human lung cancer tissue, which was stored as dropped onto the DNA card prepared according to Example 5 for three months, PCR was performed for p53 Tumor Suppressor gene. The product was cloned in a plasmid vector, and then searched again by automatic base sequencing. The method was the same as that of Example 7. The results were shown in
As the results, it was found that mutagenic p53 gene was exactly amplified (
In assaying the presence and the amount of a specific gene by a hybridization assay such as Southern blotting and the like using a complex of DNA and chitosan stored according to the method of the invention, the DNA bound to chitosan does not move in agarose gel electrophoresis, so it is difficult to conduct a blotting assay. Therefore, for the blotting assay, the DNA sample should be used with chitosan removed. Therefore, in the invention, a method has been established to remove chitosan without damaging DNA from DNA which is bound to chitosan.
As described above, the water-soluble chitosan is a cationic salt and binds to DNA, so chitosan can be isolated from DNA by treating with a strong cationic salt such as sulfate of SDS (sodium dodecyl sulfate), SOS (sodium octyl sulfate) and CTAB (cetyltrimethyl ammonium bromide). In this case, the cationic salt should be inexpensive and have no effect on the blotting assay without damaging on DNA. Therefore, in the invention, SDS (Sigma Co., USA) was used, which is inexpensive and has no effect on a hybridization reaction between DNA and probe.
A DNA/chitosan complex obtained by mixing a chitosan solution and a pancreatic cancer tissue DNA according to the method of the invention was stored at room temperature for three months. Then, 5 to 10 μg of the DNA/chitosan complex was mixed with 5% (w/v) SDS, and subjected to electrophoresis on agarose gel for Southern blotting. Southern blotting was performed for K-ras gene by a known method (Sambrook J & Russell D W, Molecular cloning: a laboratory manual, 2001). The results were shown in
From
In addition,
PCR was conducted for a human genomic DNA sample stored using the Type 1 DNA card of the invention, and then it was tested if the genotyping test is possible for it using an RFLP (restriction fragment length polymorphism) assay. The results of genotyping using the RFLP assay after PCR of the Type 1 DNA card were shown in
The present example will be described in detail below.
Using Type 1 DNA card and Type 2 DNA card and the plastic DNA ID card of the invention, the following six genes, which are associated with geriatric diseases, were prepared, respectively in a PCR tube using reaction solutions as shown in the table below.
The PCR tube containing the reaction solution, which was prepared as above, was added to a PE2700 thermal cycler (Perkin Elmer, USA), and was amplified according to each gene as follows.
1. For eNOS1/2, MTHFR1/2, AGT1/2, AT1R, ACE1 gene:
95° C./5 min, 35 Cycles (95° C./30 sec, 58° C./30 sec, 72° C./40 sec), 72° C./10 min
2. For ACE2 and APOE1/2 gene:
95° C./5 min, 35 Cycles (95° C./30 sec, 65° C./30 sec, 72° C./40 sec), 72° C./10 min
The thus-obtained PCR product of each gene was confirmed by performing electrophoresis on 1.2% agarose gel containing EtBr. The size of the product of each gene is shown in the following Table 2.
To perform RFLP with the thus-obtained PCR product, first, PCR products of other five genes (eNOS, MTHFR, AGT, AT1R and APOE) except only the ACE gene were purified using a DNA Clean & Concentrator kit (Zymo Research Corporation, CA USA) as follows.
1. To the PCR product (about 25 l), the DNA binding solution was added in double volume, i.e., 50 μl.
2. To a Zymo spin column, which had been previously provided, all of the mixture according to the above 1 was added and centrifuged at 13,000 rpm for 30 seconds.
3. The solution collected in a collecting tube was removed with a pipet.
4. 200 μl of a washing buffer was added to the column and centrifuged at 13,000 rpm for 30 seconds (repeated twice).
5. The remaining washing buffer was completely removed by centrifuging it in an empty tube at 13,000 rpm for 40 seconds. 6. The collecting tube was removed, and a clean 1.5 ml micro-centrifuge tube was provided for a new column, and then 20 μl of sterilized tertiary distilled water was added thereto, and centrifuged at 13,000 rpm for 40 seconds for elution. Alternatively, sterilized tertiary distilled water heated to 65° C. or so may be used.
For the PCR products which are purified and obtained in the above method, a restriction enzyme corresponding to each gene in the table below was prepared under the condition corresponding to each restriction enzyme. Then, they were held for reaction in a water bath at 37° C. for 4 to 6 hours, and then, the risk of each gene was searched by electrophoresis on 2.5% agarose gel.
As an application of the DNA storage method using chitosan of the invention, a PCR kit was produced wherein Taq polymerase, a primer, dNTP and a buffer and the like were added to one tube with chitosan (all in one tube), and a sample such as DNA or serum was added to the tube to lead to a PCR reaction. Characteristically, this PCR kit can be used for both of DNA storage and PCR assay.
In the present example, using DNA obtained from blood of Type B hepatitis patients, the presence of Type B hepatitis virus was searched with the PCR kit of the invention. The kit can be also applied for cancer diagnosis by diagnosis of cancer-associated gene, or diagnosis of pathogen in Type B virus hepatitis or human papilloma virus infection, sexually transmitted infection, other bacterial infections and the like.
Usage of this kit is as follows.
1 to 3 μg of a DNA template was added to an all-in-one tube containing 10 μl of 5 GG one step PCR buffer TM and hot start type Taq polymerase (AmpliTaq Gold, PerkinElmer, USA), 2.0 μl of 10 mM dNTP mixture, 1.0 μl of each of forward primer and reverse primer (5 pmol) of the target gene, and a 0.1% concentration of chitosan. Distilled water was filled into the kit to a total volume of 50 l, and well mixed, and then PCR was performed. First, reverse transcription reaction was performed at 50° C. for 30 minutes, denaturation was conducted at 95° C. for 15 minutes, followed by a total of 25 to 40 cycles of 94° C. for one minute, 53° C. for 40 seconds and 72° C. for one minute, then PCR amplification was completed with the reaction of 72° C. for 10 minutes. As a result, the presence of DNA of Type B hepatitis virus was found using the PCR kit of the invention (no data attached).
1. Receiving transported personal sample such as blood or oral cell, DNA card and the like, and personal photograph data (S201).
2. Isolation of genomic DNA from the sample and test of blood typing (S202).
3. Assay of HLA genotype using the extracted genomic DNA, test of STR personal identification gene and analysis for results (S203).
4. Incorporation of a paper DNA card for attachment with the plastic card, and preparation of the frame of the DNA ID card by adding heat and pressure (S205).
5. Dropping of personal DNA or blood on the plastic DNA ID card and the card for storage, which are produced in S204 (S205). Dropping of the DNA or blood on the plastic DNA ID card is conducted by injecting DNA or blood into the paper DNA card through the hole of the PVC core layer penetrating the PVC soft layer using a needle in. After the injection, the hole caused by the needle is blocked by hologram, sticker and the like.
6. Encoding of the analyzed data on the magnetic bar or the chip of the plastic DNA ID card (S206). The magnetic bar is usually constituted by three tracks. The tract on which English can be written is the first track, which has 78 bits available. The second track has 38 bits available, and the third track has 107 bits available. Therefore, as shown in
7. Transcription of personal photograph and brief personal information to carve them on the plastic DNA ID card by the embossing method (S207).
8. Input of personal data into a super computer (for server), assigning of specific personal ID code to the plastic DNA ID card, and setting personal password (S208).
9. Construction and Operation of POS system to allow a person to access information according to the conditions for security maintenance using an ID and a password, which are recorded in the plastic DNA ID card both at home and abroad if necessary by each person, hospital, each organization or bank and the like (S209).
It was ascertained if gene can be amplified from the DNA which was stored in Type 1 or Type 2 paper DNA card, which was incorporated into a plastic DNA ID card, which has been made previously by treatment at high temperature and high pressure, by amplifying eNOS gene, which is one of geriatric disease genes, by PCR. The method was performed in the same manner as in the above Example 10.
Type 1 DNA card and Type 2 DNA card were incorporated, respectively into a plastic DNA ID card which had been prepared at high temperature and high pressure, to have human genomic DNA and blood stored as submerged in each of them. Then, using these plastic DNA ID cards, eNOS gene, which is one of geriatric disease genes, was amplified by PCR for comparison. The results are shown in
Type 1 DNA card for DNA dropping and Type 2 DNA card for blood dropping were attached, respectively on a plastic card, which was prepared at high temperature and high pressure to prepare a DNA ID card. To find appropriate concentration and amount of samples to be dropped on each of them, in case of genomic DNA, DNA sample was dropped in 150 ng/? concentration in various volumes using the card fragment preparation as above, and eNOS gene was amplified in PCR and assayed. The results are shown in
The difference was compared and analyzed by amplifying eNOS gene, which is one of geriatric disease genes by PCR, between the case that the card fragment is treated with a washing buffer as a pretreatment process and the case that it is not treated in PCR amplification of the sample stored in a plastic DNA card prepared at high temperature and high pressure. The results are shown in
The DNA storing method using the water-soluble chitosan according to the present invention, is excellent in DNA storage efficacy, and makes it possible to store DNA stably at room temperature for a long time on the contrary to the conventional method. Using this method, it is possible to store and carry not only small plasmid DNA, but also genomic DNA and cDNA of big plants and animals, and genomic DNA of fungi, bacteria and virus at room temperature stably for a long time. DNA stored according to the present method is useful for all of gene assays such as PCR and RFLP, cloning, base sequencing, Southern blotting and the like. Furthermore, it can be used broadly in a PCR kit and the like, and it is simple and economical. Especially, the Type 1 DNA card of the invention is very useful for storing and carrying multiple DNA samples at room temperature for a long time, and Type 2 DNA card can store various bio-samples containing DNA such as blood without damage to DNA for a long time at room temperature. In addition, for the sample stored in these DNA cards, various gene assays such as PCR and PCR-RFLP, hybridization, base sequencing, SNP assay and the like can be performed accurately and simply even after a long time, which is very helpful in basic researches and also various clinical treatments. In addition, the DNA ID card of the invention can play a role of DNA bank which stores personal DNAs, and it is also helpful for personal identification and medical treatment such organ transplant and the like by storing the personal gene information as well.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1020040071254 | Sep 2004 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR05/00774 | 3/18/2005 | WO | 3/6/2007 |
