TW201520333A - Method for preserving deoxyribonucleic acid (DNA) - Google Patents

Abstract

The present invention provides a method for preserving deoxyribonucleic acid (DNA), comprising: preparing a collection rod with a collection part having a smooth surface, the collection part being attached with deoxyribonucleic acid (DNA); placing the collection rod in a preservation bottle containing buffer solution to prolog the preservation time of deoxyribonucleic acid (DNA), wherein the buffer solution contains 20 mM to 60 mM of ethylenediamine tetraacetic acid (EDTA). The method for preserving deoxyribonucleic acid of this invention uses the ethylenediamine tetraacetic acid of a specific concentration range to avoid degradation of deoxyribonucleic acid; in addition, the selective applicability of the collection rod and the buffer solution may also prolong the preservation time of deoxyribonucleic acid so as to increase the test accuracy.

Description

Method of preserving deoxyribonucleic acid

The present invention relates to a method for preserving deoxyribonucleic acid, and more particularly to a method for placing a gene collection rod containing deoxyribonucleic acid in a preservation flask to prolong the storage time of the deoxyribonucleic acid.

A common method of gene collection is to collect the DNA of the subject with a collection rod, and then put the collection rod of the subject DNA into an empty storage bottle or save with a desiccant. bottle.

Because during the transportation process, the collection rod with the subject tissue will collide with the bottle body and affect the collection concentration, and the dry storage of the deoxyribonucleic acid will deoxyribonucleic acid degradation and affect the accuracy of subsequent detection. .

In addition, the affinity between the collection rod and the sample collected affects the amount of DNA dissolved in the subsequent treatment solution. For example, when the adhesion of the sample to the collection rod is greater than the interaction between the sample and the subsequent treatment solution, a large amount of sample will remain on the collection rod; and the material of the collection rod itself and the specific chemical functional group of the subsequent treatment solution It may even affect the degree of degradation of the sample, which in turn affects the quality of the DNA in the sample. Thus, there is a technical need in the art to stably preserve the DNA taken by a subject.

In view of the prior art method of preserving DNA is easy The invention degrades the DNA to affect the accuracy of the subsequent detection, and the like, and an object of the present invention is to provide a method for preserving the DNA contained in the sample taken by the physical method through a suitable collection rod and The selectivity of the buffer is suitable to extend the storage time of the DNA to improve the accuracy of the detection.

In order to achieve the above object, the present invention provides a method for preserving deoxyribonucleic acid, comprising: collecting a collection rod having a smooth surface collection portion, wherein the collection portion is dipped with a subject's deoxyribonucleic acid; The collection rod is placed in a buffer containing buffer to prolong the storage time of the DNA, wherein the buffer contains ethylenediamine tetraacetic acid (concentration) at a concentration of 20 millimolar (mM) to 60 mM. EDTA).

According to the present invention, the "smooth surface" is made of a porous material as described herein. Preferably, the collecting portion of the collecting rod is formed by a thermoplastic plastic foaming process, wherein the thermoplastic plastic is selected from the group consisting of nylon (nylon), polyethylene (polyethylene, PE). ), a group of polypropylene (PP), polyurethane (PU), polystyrene (PS), polyesters, and combinations thereof.

More preferably, the collecting portion of the collecting rod has a cylindrical shape, a cone shape, a water drop shape or a spherical surface.

Preferably, the buffer further comprises a sodium chloride selected from the group consisting of sodium chloride a group consisting of (NaCl), 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride, Tris-HCl, and sodium dodecyl sulfate Group of substances.

More preferably, the buffer is in the total concentration of the buffer. Based on the degree, the concentration of sodium chloride is between 20 mM and 150 mM, the tris hydroxy-aminomethane-hydrochloric acid is weakly alkaline, and the concentration is from 10 mM to 60 mM; and based on the total volume of the buffer, the dodecyl sulphate The concentration of sodium is between 0.1 w/v% and 10 w/v%.

Preferably, the buffer includes, but is not limited to, sulphur Guanidine isothiocyanate solution (GIT solution), guanidine thiocyanate solution (GT solution), GST solution (purchased from Geneaid).

The method for preserving deoxyribonucleic acid of the present invention is A concentration range of ethylenediaminetetraacetic acid to avoid deoxyribonucleic acid degradation; and because the material of the collection part is made of a smooth surface composed of a porous material, the surface area of the collection part can be increased to obtain more test. The deoxyribonucleic acid can also be easily collected and can reduce the discomfort of the subject; in addition, the adhesion of the sample to the collection rod can be reduced by the components in the buffer, so that a larger amount of the sample can be separated from the collection rod. And entering the buffer, thereby increasing the concentration of deoxyribonucleic acid obtained by subsequent processing.

The lanes 1, 2, and 3 shown in the respective figures are nylon brush sets with a diameter of 4, 5, 6 are foamed nylon swabs, and 1, 4 are untreated groups; diameters 2 and 5 are collections of collection rods using prior art desiccant; diameters 3 and 6 are collections of GST buffers. In the collection part, M is a DNA molecular marker, wherein the arrow indicates that the DNA is degraded [ie, smear].

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing the deoxyribonucleic acid agarose gel of sample 1 which was placed at room temperature for 2 days after collecting a sample of a subject with a nylon brush and a foamed nylon swab, respectively.

Fig. 2 is a diagram showing the deoxyribonucleic acid agarose gel electrophoresis of sample 2 which was placed at a low temperature for 4 days after collecting a sample of a subject with a nylon brush and a foamed nylon swab according to Example 1 of the present invention.

Fig. 3 is a diagram showing the deoxyribonucleic acid agarose electrophoresis pattern of sample 3 which was placed at room temperature for 8 days after collecting a sample of a subject with a nylon brush and a foamed nylon swab, respectively, in Example 1 of the present invention.

In order to understand the technical features of the present invention in detail, and in accordance with the contents of the specification, the following preferred embodiments are further described in detail below. The present invention provides a method for preserving deoxyribonucleic acid, which comprises: having one a collecting rod of the collecting portion of the smooth surface, wherein the collecting portion is dipped with the DNA of the subject; and the collecting rod is placed in a storage bottle containing the buffer to prolong the storage time of the DNA, wherein The buffer contains ethylenediamine tetraacetic acid (EDTA) at a concentration ranging from 20 millimolar (mM) to 60 mM.

In a particular embodiment, the collection portion is cylindrical and has The utility model has the effect of reducing the discomfort of the subject, the collecting part has an elevated surface area, and can obtain more DNA of the subject; and the material of the collecting part is a foamed nylon which is foamed. And through the collection department, the oral mucosa cells of the subject can be collected, and the discomfort of the subject can be minimized, and the number of samples collected can be improved for accurate detection.

The size of the storage bottle is at least capable of accommodating the collection of the collection rod In a specific embodiment, the buffer is a GST buffer (purchased from Geneaid) and is based on the total concentration of the buffer. The buffer contains 25 mM ethylenediaminetetraacetic acid, a concentration of 100 mM sodium chloride, and a concentration. It was 10 mM and pH 8.0 trishydroxymethylaminomethane-hydrochloric acid and a concentration of 0.5 w/v% sodium lauryl sulfate.

In a preferred embodiment of the invention, the buffer is 胍 A guanidine isothiocyanate solution (GIT solution) comprising 4 M guanidinium thiocyanate, 50 mM, pH 7.5 Tris-HCl, and 25 mM ethylenediaminetetraacetic acid.

In the following examples, the collection rod used includes a nylon brush (nylon brush) (purchased from Airway, model 30021) and foamed tip swab (purchased from Airway, model number 30221); The longitudinal length of the portion is 20 mm (mm) and the width is 7 mm, and the collecting portion has a plurality of bristles arranged equidistantly from each other and perpendicular to the collecting rod; wherein the collecting portion of the foamed nylon swab is The longitudinal length is 22 mm and the width is 7 mm, and the collecting portion is made of a porous material formed by a foaming process.

Example 1 Collection type, temperature and time of the collection part of the collection rod Effect on the concentration of deoxyribonucleic acid

After collecting the oral mucosa of the subject several times (preferably 6 to 7 times), the collection part containing the subject's DNA is separately placed in a desiccant (desiccant, purchased) In the container of isohelix), the storage bottle containing buffer or not treated at all (as a control), and the above container, storage bottle or control group is placed at low temperature (4 ° C), room temperature (25 ° C) and different The number of days (2 days, 4 days, 8 days) was placed, and the DNA was extracted by the following method.

The collection portion containing the subject's DNA was placed in a microcentrifuge tube containing 500 μl of GST buffer and 20 μL of proteinase k in a microcentrifuge tube at 60 ° C for 10 minutes. The collection part was taken out, and 500 μL of GST buffer was added thereto and shaken uniformly at 60 ° C for 10 minutes to dissolve the cell membrane and the nucleus. After 500 μL of absolute ethanol was evenly shaken, the above solution was taken and placed on a GD column (GD column, purchased from Geneaid), centrifuged at 14,000 x g to 16,000 x g for 1 minute, and then 100 μL was added. Deoxyribonucleic acid can be obtained by centrifugation of the elued buffer or TE buffer again at a rate of 14,000 x g to 16,000 x g for 30 seconds.

The above method for extracting deoxyribonucleic acid is the preferred embodiment provided by the present invention, and those skilled in the art can convert, modify and change the method for extracting deoxyribonucleic acid according to the usual knowledge at the time, therefore, the above The method of extracting deoxyribonucleic acid is used as an illustration and not a limitation.

Deoxyribonucleic acid samples obtained in the above steps were respectively Deoxyribonucleic acid concentration analysis was performed with ethidium bromide (EtBr), and deoxyribonucleic acid purity analysis was performed at a ratio of absorbance at 260 nm (nm) to 280 nm.

The ethidium bromide quantification method is as follows: a known concentration of a DNA molecular weight standard (DNA marker) and an unknown concentration of a DNA sample obtained in the above step are subjected to agarose gel electrophoresis. After being stained with ethidium bromide, photographing on a UV light box to compare the brightness of the standard concentration with the unknown concentration of deoxyribonucleic acid, thereby calculating the concentration, purity and degradation degree of the deoxyribonucleic acid; The purity of oligoribonucleic acid is the ratio of the absorbance of the sample at 260 nm to the absorbance at 280 nm. The purity of the DNA is determined by the numerical value. When the OD ₂₆₀ /OD ₂₈₀ = 1.8, the purity of the DNA is high. Less than 1.8 means that the purity of the deoxyribonucleic acid is low; wherein the degree of degradation of the deoxyribonucleic acid is quantified by using the Image J software to agarose containing the DNA sample, and each sample is calculated to be intact and not decomposed. The ratio of the pixel value of the oligoribonucleic acid [ie, the major] to the pixel value of the deoxyribonucleic acid [ie, smear], the higher the ratio, the saved Deoxyribonucleic acid more complete.

1: The collecting portion has a plurality of bristles arranged equidistantly from each other and perpendicular to the collecting rod, so that the surface of the collecting portion is irregular.

2: The collecting part is made of a porous material formed by a nylon foaming process, so that the surface of the collecting part is smooth and has a cylindrical shape or a conical shape.

3: Untreated means that after collection by the collection part, it is not directly dried in the desiccant or GST buffer, and is directly placed at the above specific temperature or number of days before extraction to obtain deoxyribonucleic acid.

When the collection part is placed at room temperature (25 ° C), extracting deoxygenation after 2 days As shown in Figure 1 and Table 1, although the nylon brush can obtain a higher concentration of deoxyribonucleic acid in the untreated group, the foamed nylon swab group is either untreated, desiccant or placed in GST buffer. The DNA obtained from the preserved group has a higher concentration.

When the collection part is placed at low temperature (4 ° C), extract the deoxygenate after 4 days. In the case of the sugar nucleic acid, as shown in Fig. 2, when the collection portion of the collection rod is placed in the untreated and desiccant group, the deoxyribonucleic acid extracted by the desiccant group is degraded.

When the collection part is placed at room temperature (25 ° C), extracting deoxygenation after 8 days In the case of ribonucleic acid, as shown in Fig. 3, when the extraction portion of the collection rod is untreated, the extracted DNA is degraded by either a nylon brush or a foamed nylon swab.

In summary, when stored at low temperature (4 ° C), avoid deoxygenation Ribonucleic acid degradation to prolong the storage time of DNA; overall, foamed nylon swabs can be harvested to obtain higher concentrations of deoxyribonucleic acid than nylon brushes; and as shown in Table 2, foamed nylon wipes The sub-column with GST buffer is more complete than the deoxyribonucleic acid obtained from the foamed nylon swab in combination with the desiccant or the untreated group.

Claims (6)

A method for preserving deoxyribonucleic acid, comprising: collecting a collection rod having a smooth surface collection portion, wherein the collection portion is stained with deoxyribonucleic acid; and placing the collection rod in a storage bottle containing buffer to extend The storage time of the oligoribonucleic acid, wherein the buffer contains ethylenediamine tetraacetic acid (EDTA) at a concentration of from 20 millimolar (mM) to 60 mM. The method of claim 1, wherein the collecting portion of the collecting rod is formed by a thermoplastic plastic foaming process, wherein the thermoplastic plastic is selected from the group consisting of nylon (nylon), polyethylene (polyethylene, PE). ), a group of polypropylene (PP), polyurethane (PU), polystyrene (PS), polyesters, and combinations thereof. The method of claim 1, wherein the collecting portion of the collecting rod is cylindrical, conical, drop-shaped or has a spherical surface. The method of any one of claims 1 to 3, wherein the buffer further comprises selected from the group consisting of sodium chloride (NaCl), trimethylolamine-hydrochloride [2-amino-2-(hydroxymethyl)-1 , a group of 3-propanediol hydrochloride, Tris-HCl] and sodium dodecyl sulfate. The method of claim 4, wherein the buffer comprises sodium chloride at a concentration between 20 mM and 150 mM, and tris (hydroxymethylaminomethane-hydrochloric acid) at a concentration between 10 mM and 60 mM; the concentration is between 0.1 w/v% and 10 w/v% sodium lauryl sulfate. The method of claim 4, wherein the collecting portion is made of foamed nylon.