JP2007020440A - Method for separation of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid separation means for the easy and safe separation of nucleic acid, enabling easy automation and applicable to a relatively small-sized apparatus, etc. <P>SOLUTION: The invention provides a method for separating nucleic acid from a biological specimen by treating the biological specimen with a cationic surfactant and performing electrophoresis. The nucleic acid is retained in a prescribed region and proteins and lipids are moved to the cathode side by the electrophoresis by carrying out the electrophoresis after the treatment of the biological specimen with a cationic surfactant. Accordingly, nucleic acids can be separated from a biological specimen containing proteins and/or lipids. The invention can be applied to a gene detection method to use DNA chips, etc. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid separation method. More particularly, the present invention relates to a nucleic acid separation method for separating a nucleic acid from a biological sample by performing electrophoresis after treating the biological sample with a cationic surfactant.

With recent progress in genome analysis, attempts have been made to introduce genetic testing into medical fields.
Genetic testing is testing and analysis performed on nucleic acids collected from biological samples. Genetic testing may be able to detect hereditary diseases and their risk, infectious diseases (pathogenic microorganisms), and malignant tumors with high accuracy.

  For example, when blood or tissue is collected from a human body and subjected to genetic testing, it is necessary to separate and purify only nucleic acid from a biological sample such as blood.

As methods for separating and purifying nucleic acids, for example, silica adsorption method, alkali-SDS method, AGPC method and the like are known.
The silica adsorption method is a method in which nucleic acid is adsorbed on silica and separated. This method utilizes the property that nucleic acids are selectively adsorbed on the silica surface in the presence of chaotropic ions.
The alkali-SDS method is a method used for extraction of plasmid DNA and the like. First, the nucleic acid is denatured into a single strand by treatment with an alkaline solution containing SDS, and then sodium acetate or the like is added to precipitate protein SDS complex or polymer RNA. Since plasmid DNA has a closed circular structure, it remains in solution without being denatured.
The AGPC (Acid Guanidinium-Phenol-Chloroform) method is a method used for RNA extraction. By performing phenol extraction under acidic conditions, proteins can be removed and RNA can be efficiently recovered.

As prior literature, for example, Patent Document 1 discloses a nucleic acid purification method using silica, Patent Document 2 discloses a nucleic acid separation method utilizing two-phase partitioning, and Patent Document 3 discloses a cation. Nucleic acid separation methods using surfactants are each described.
JP 2005-110503 A JP 2003-339379 A Japanese Patent Laid-Open No. 2-96630

  Conventional nucleic acid separation methods have complicated procedures and operations, and it is often necessary to use a relatively large apparatus such as centrifugation during the procedure. For this reason, it is difficult to automate procedures and operations, and it is difficult to apply to a relatively small device.

  In addition, since conventional nucleic acid separation methods often use harmful substances such as organic solvents and chaotropic salts during the procedure, there are problems in using these methods in medical settings from the viewpoint of safety. there were.

  Therefore, the main object of the present invention is to provide a nucleic acid separation means that can be carried out simply and safely, is easily automated, and can be applied to a relatively small apparatus.

  The present invention provides a nucleic acid separation method for separating a nucleic acid from a biological sample by performing electrophoresis after treating the biological sample with a cationic surfactant.

  By treating the biological sample with a cationic surfactant, the negative charge of the nucleic acid present in the biological sample can be neutralized, and the protein present in the biological sample can also be charged to a positive charge. . Moreover, the lipid which exists in a biological sample can be emulsified and the micelle with a positive charge can be formed.

  Therefore, by treating a biological sample with a cationic surfactant and then performing electrophoresis, when electrophoresis is performed, the nucleic acid can remain in a predetermined region and proteins and lipids can be moved to the cathode side. it can. That is, nucleic acids can be separated from biological samples containing proteins and / or lipids.

  The nucleic acid separation method according to the present invention is relatively simple in procedure and operation, and uses a cationic surfactant, so that it is highly safe. In addition, since this method can perform nucleic acid separation as long as it has electrophoresis means, the apparatus can be miniaturized. In addition, this nucleic acid separation method can be applied to a nucleic acid detection method using, for example, a DNA chip.

  According to the present invention, nucleic acids can be separated from biological samples and the like simply and relatively safely. Moreover, the nucleic acid separation method according to the present invention has advantages that it is easy to automate and can be applied to relatively small devices.

<Nucleic acid separation method according to the present invention>
In this nucleic acid separation method, a biological sample is treated with a cationic surfactant and then subjected to electrophoresis to separate nucleic acids from the biological sample.
That is, this method includes at least three procedures: (1) a procedure for collecting and preparing a biological sample, (2) a procedure for treating the biological sample with a cationic surfactant, and (3) a procedure for performing electrophoresis. Including.
Hereinafter, it demonstrates in order.

(1) Procedure for collecting and preparing biological samples:
First, according to the purpose, a biological sample is collected from a human body or the like, and in some cases, the collected biological sample is prepared.

  The biological sample only needs to contain nucleic acid. For example, a biological sample such as blood, body fluid, tissue, or cell may be collected from a human body, or a cultured cell or the like may be used as a biological sample according to the purpose.

In the present invention, since cells can be lysed when the biological sample is treated with a cationic surfactant in the next step, in particular, as a pretreatment, cells are lysed to prepare a cell lysate. There is no need to do so. That is, the present invention has an advantage that it is not necessary to perform a complicated biological sample preparation operation.
However, the nucleic acid separation method according to the present invention can also be applied to a prepared biological sample. That is, for example, when separating nucleic acid from cell lysate, frozen cells, etc., the nucleic acid separation method according to the present invention can be applied.

(2) Procedure for treating a biological sample with a cationic surfactant:
Next, the collected and prepared biological sample is treated with a cationic surfactant.

The cationic surfactant is not particularly limited as long as it has an adsorptive power to proteins.
For example, DTAB (Dodecyl Trimethyl Ammonium Bromide), Cetyl trimethyl ammonium bromide, Lauryl trimethyl ammonium chloride, Steady Trim Trim.

By adding a cationic surfactant to the collected and prepared biological sample and allowing it to stand for a predetermined time, the cells are lysed, the nucleic acid present in the biological sample is neutralized, and the protein and lipid are positively charged. have.
In this procedure, the mixture of the cationic surfactant and the biological sample may be appropriately stirred, or the mixture may be heat-treated for a predetermined time.

(3) Procedure for performing electrophoresis:
Next, a direct current electric field is applied to the mixture of the cationic surfactant and the biological sample to perform electrophoresis.

  By treating a biological sample with a cationic surfactant, nucleic acids present in the biological sample are neutralized, and proteins and lipids have a positive charge. Therefore, the nucleic acid hardly moves even by electrophoresis and stays in a predetermined region, while the protein and lipid move to the cathode side by electrophoresis. Thus, nucleic acids can be separated from biological samples containing proteins and / or lipids.

The nucleic acid separation method according to the present invention can be automated in a biological sample supply means, a cationic surfactant supply means, an electrophoresis means, and an apparatus having a predetermined reaction region.
That is, for example, a step of supplying a biological sample to a predetermined reaction region, a step of supplying a cationic surfactant to a predetermined reaction region, a step of performing electrophoresis by applying a DC electric field, and the above three steps are programmed in advance. It can be automated within the device.

<Gene detection method using DNA chip>
In this gene detection method, at least the following procedures (1) to (3) are performed.
(1) Procedure for supplying a biological sample and a cationic surfactant to each well,
(2) A procedure for performing electrophoresis in each well to separate nucleic acid from a biological sample,
(3) A procedure for detecting hybridization between a nucleic acid for detection held or fixed in a well and a nucleic acid present in a biological sample.
Hereinafter, a description will be given with reference to FIG.

  Each drawing of FIG. 1 is a schematic cross-sectional view of one of a plurality of wells provided on the substrate surface of the DNA chip. The well 1 includes a reaction region 2 serving as a hybridization field, a detection nucleic acid 3 immobilized on the reaction region, and an electrode 4 (a positive electrode 41 and a negative electrode 42 provided so as to be able to perform electrophoresis). ).

  FIG. 1A shows a procedure for supplying the biological sample S and the cationic surfactant I to the well 1.

In FIG. 1A, the biological sample S and the cationic surfactant I are supplied to the well 1 from the biological sample supply means 5 and the cationic surfactant supply means 6, respectively (arrows X1, X2), The reaction region 2 is filled with a mixed solution F of the biological sample S and the cationic surfactant I. The mixed solution F contains nucleic acid 7, protein 8, lipid 9, and the like.
In this operation, the biological sample S and the cationic surfactant I may be mixed (or mixed and prepared) and then supplied to the well 1. In that case, the supply means can be made one.

  FIG. 1B shows a procedure for performing electrophoresis in the well 1 to separate the nucleic acid 7 from the biological sample S.

  Electrophoresis is performed by applying a DC electric field between the plus electrode 41 and the minus electrode 42. By performing electrophoresis, the nucleic acid 7 stays in the vicinity of the detection nucleic acid 3, but the protein 8 and the lipid 9 have a positive charge due to adsorption of the cationic surfactant, and thus move toward the negative electrode 42. (Arrows X3, X4). Thereby, the nucleic acid 7, protein 8, lipid 9, etc. can be isolate | separated.

  FIG. 1C shows a procedure for detecting the hybridization between the detection nucleic acid 3 held or fixed in the well 1 and the nucleic acid 7 present in the biological sample S.

  Since impurities such as protein 8 and lipid 9 can be removed from the vicinity of the detection nucleic acid 3 by electrophoresis, noise can be reduced and hybridization can be detected with high accuracy.

The gene detection method according to the present invention includes a biological sample supply means, a cationic surfactant supply means, an electrolytic application means for applying a DC electric field between the electrodes of the DNA chip, and a hybridization detection means. Automatic processing can be performed in a hybridization detection apparatus or the like.
That is, a step of supplying a biological sample from a biological sample and / or a cationic surfactant supply means to a predetermined reaction region, a step of applying a DC electric field to a DNA chip for electrophoresis, a hybridization in the predetermined reaction region The above-described three steps can be automated in a DNA chip hybridization detection apparatus or the like by describing them in advance with a program.

In addition, the gene detection method according to the present invention has the following advantages.
First, the procedure from supplying the biological sample S or the like to the well 1 (the procedure in FIG. 1A) to the procedure for detecting hybridization or the like (the procedure in FIG. 1C) is performed in one well. Therefore, the operations performed in each of these procedures can be performed continuously with one apparatus, and can be easily automated.
In addition, by using a DNA chip, etc., a series of steps can be carried out continuously and automatically, so that various and large numbers of genetic tests can be performed in a single assay relatively easily. Can do.

  In Example 1, it was examined whether or not the nucleic acid and the protein can be separated by adding a cationic surfactant to the mixed solution of the nucleic acid and the protein.

In this experiment, BSA (Bovine Serum Albamin, hereinafter the same) is used as a protein sample, plasmid DNA subjected to restriction enzyme treatment is used as a nucleic acid sample, and DTAB (DodecylTrimethylAmmonium Bromine, hereinafter the same) is used as a cationic surfactant. It was.
As a control, SDS (Sodium Dodecyl Sulfate, hereinafter the same), which is an anionic surfactant, was used.

The outline of the experimental procedure is as follows.
First, appropriate amounts of plasmid DNA and BSA were dissolved in 50 mM Tris-HCl (pH 6.8). Next, DTAB was added to a final concentration of 2%, and heat denaturation treatment was performed at 100 ° C. for 3 minutes. Next, glycerol was added to a final concentration of 10%.
Next, a sample was placed in each well of a 1% agarose gel, and electrophoresis was performed at 50 V for 45 minutes. TAE (Tris Acetate EDTA) was used as the electrophoresis buffer.
Next, the agarose gel was collected and stained with ethidium bromide and then photographed with a UV transilluminator to observe the behavior of the nucleic acid. Moreover, about the same agarose gel, after performing CBB dyeing | staining, the behavior of protein was observed by visible light observation.

The results are shown in FIG.
In the figure, the photograph on the right is a photograph when ethidium bromide staining is performed, and the photograph on the left is a photograph when CBB staining is performed. In both photographs, the right side is the anode and the left side is the cathode.
In both photographs, the uppermost lane and the second lane from the top are controls, the uppermost lane is an electrophoresis of nucleic acid markers, and the second lane is an electrophoresis of protein markers.
In both photographs, the third to fifth lanes from the top are those for electrophoresis of protein (BSA), the third lane from the top is for electrophoresis of BSA alone, and the fourth lanes are for BSA. SDS was added to SDS, and the fifth lane was electrophoresed by adding DTAB to BSA.
In both photographs, the sixth to eighth lanes from the top are those obtained by electrophoresis of nucleic acid (plasmid DNA), the sixth lane from the top is obtained by electrophoresis of plasmid DNA alone, and the seventh lane is SDS is added to plasmid DNA and electrophoresed, and the eighth lane is electrophoresed by adding DTAB to plasmid DNA.
In both photographs, the ninth to tenth lanes from the top are obtained by electrophoresis of nucleic acid (plasmid DNA) and protein (BSA), and the ninth lane from the top is obtained by electrophoresis of plasmid DNA and BSA. The tenth lane is obtained by adding SDS to plasmid DNA and BSA, and the tenth lane is obtained by adding DTAB to plasmid DNA and BSA.

In the third to fifth lanes from the left in the photograph of FIG. 2, when BSA alone is carried out by electrophoresis, BSA moves to the right side (anode side) (third lane from the top). When SDS was added to BSA, BSA further moved to the right side (anode side) (also the fourth lane), whereas when DTAB was added to BSA, BSA moved to the left side (cathode side) (also 5 The second lane).
These results indicate that the protein can be moved to the cathode side when electrophoresis is performed by treating the protein with a cationic surfactant.

In addition, in the sixth to eighth lanes from the top of the photograph on the right side of FIG. 2, by performing electrophoresis, when SDS was added to plasmid DNA alone or to plasmid DNA, the DNA moved to the right (upper side) When the DTAB was added to the plasmid DNA, the DNA stayed in the well and did not migrate (also the eighth lane).
These results indicate that by treating the nucleic acid with a cationic surfactant, the nucleic acid can remain without moving even when electrophoresis is performed.

In addition, in the ninth to tenth lanes from the upper right photograph in FIG. 2, the same result as above was obtained when both BSA and plasmid DNA were mixed.
Therefore, the above experimental results indicate that nucleic acid and protein can be separated by treating a mixture of both nucleic acid and protein with a cationic surfactant and performing electrophoresis.

According to the present invention, since nucleic acids can be separated from a biological sample with simple and few operations, genetic testing and the like may be more easily performed.
For example, it may be possible to detect a hereditary disease and its risk by collecting predetermined cells (including blood) from a human body and separating nucleic acids from the cells. Further, by collecting predetermined cells from diseased animals and detecting the nucleic acid of the pathogenic microorganism contained in the biological sample, the type and origin of the pathogenic microorganism may be detected with high accuracy. Similarly, by collecting cancer cells and isolating nucleic acids contained in the cancer cells, it is possible to detect tumor types and malignancy, and to determine applicable anticancer agents with high accuracy. Could be possible.

In addition, the present invention is applicable to gene analysis using a DNA chip or the like.
For example, an electrode is provided in each of a plurality of wells formed on the surface of a substrate such as a DNA chip so that electrophoresis can be performed. Then, supplying biological samples to each well, performing electrophoresis, keeping the nucleic acid in a predetermined region in the well, moving the protein to the cathode side, and then proceeding with a reaction such as hybridization, Gene detection accuracy can be increased.

FIG. 4 is a schematic cross-sectional view showing one of a plurality of wells provided on the substrate surface of the DNA chip, and showing the operation procedure of the gene detection method according to the present invention. The drawing substitute photograph which shows the electrophoresis image at the time of performing electrophoresis after adding a cationic surfactant to the mixed solution of a nucleic acid and protein.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Well formed in substrate surface of DNA chip 2 Reaction region 3 Nucleic acid for detection 4 Electrode 41 Positive electrode 42 Negative electrode 5 Biological sample supply means 6 Cationic surfactant supply means 7 Nucleic acid 8 (in biological sample) 8 (Biological substance) Protein in sample 9 Lipid in biological sample F Mixture of biological sample S and cationic surfactant I I Cationic surfactant S Biological sample

Claims (4)

  A nucleic acid separation method for separating a nucleic acid from a biological sample by performing electrophoresis after treating the biological sample with a cationic surfactant.   The nucleic acid separation method according to claim 1, wherein the nucleic acid is separated from a biological sample containing protein and / or lipid. 2. The method for separating nucleic acids according to claim 1, wherein when electrophoresis is performed, the nucleic acids are kept in a predetermined region and the proteins are moved to the cathode side.
A plurality of wells are provided on the surface of the substrate, and a DNA chip having electrodes provided on the wells is used.
A nucleic acid detection method for detecting a specific nucleic acid present in a biological sample by performing at least the following procedures (1) to (3).
(1) Procedure for supplying a biological sample and a cationic surfactant to each well,
(2) In each well, a procedure for separating nucleic acid from a biological sample by performing electrophoresis by applying a direct current electric field between the electrodes,
(3) A procedure for detecting hybridization between a nucleic acid for detection held or fixed in a well and a nucleic acid present in a biological sample.