JPH02268682A - Preparation of dna specimen - Google Patents

Abstract

PURPOSE:To reduce an amount of DNA adsorption and to efficiently reacting a very small amount of DNA by pouring a substance to be adsorbed on a wall of container without damaging a reaction system. CONSTITUTION:A substance (DNA or polyoxyethylene sorbitan monolaurate, nonionic surfactant) to be adsorbed on a wall of container without damaging a reaction system is poured, a substance except DNA of specimen is adsorbed on the wall of container, an amount of DNA of specimen adsorbed on the wall of container is reduced so that reaction ratio can be improved.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for preparing a DNA sample, and particularly to a method suitable for efficiently reacting sensitive DNA in an automatic device.

[traffic technology]

Taking the base sequencing process as an example, conventional methods are based on Takara Shuzo Co., Ltd.'s M13 Sequencing Kit Instruction Manual (19
pmole (pmole) as described in
) order template DNA, equimolar amounts of primers, buffer, and distilled water are mixed and heated to approximately 60°C to proceed with the primer annealing reaction, and a mixture of DNA polymerase and dNTP/ddNTP is added to the solution to perform complementation. I have been performing chain synthesis reactions. That is, no particular measures have been taken to prevent adsorption of DNA samples.

[Problem to be solved by the invention]

In the conventional method, there was a problem in that when the amount of DNA-M became low, the DNA was adsorbed to the reaction container and the reaction rate decreased. A reaction vessel made of plastic such as polypropylene is used because it is cheap, lightweight, and easy to handle, and the adsorption amount is negligible when there is a sufficient amount of DNA. However, when the absolute amount of DNA decreases, the reaction rate decreases due to the effect of DNA adsorption to the plastic wall. The results of the study of the reaction rate in the base sequencing process are shown below.

Figure 3 shows standard conditions for template DNA of 0.5 PIIO.
This figure shows a comparison of one NA fragment spectrum for 16 (Figure b) and its half, 0 and 25 pmole (Figure 8). 2 The horizontal axis for each run is electrophoresis time, and the vertical axis is the relative value of fluorescence intensity. The numbers in the middle indicate the base length of the N A fragment. 6 When template DNA 1 is halved, the S/N of the peak representing the DNA fragment is 0.52 mo1 on average. It has decreased to about 20% of when the mold e was supplied.

It became difficult to identify the reaction peak.

Figure 4 summarizes the above-mentioned trends using the reaction rate as an indicator. DNA
The b ratio of 0 and 4 pmole was set to 1. 0.4
When the amount of template DNAff is larger than pmole, the reaction rate increases slowly, but when the amount of template 1) NA is smaller than 0,4 pmole, the effect of the DNA adsorption phenomenon becomes stronger and the reaction rate decreases rapidly. Here, we used the base sequencing process as an example, but DNA adsorption is
This is a problem when reacting with DNA.

The NA sample preparation process requires only one burden on the operator.
(There is a strong demand for automation, and it is expected that automation will progress in the future. Therefore, it is necessary to adopt a method that is easy to automate in the adsorption rainproofing method as well.

The present invention reduces DNA absorption and reduces the amount of DNA absorbed. 1) NA
[Means for Solving the Problems] In order to achieve the above object, the present invention aims to provide a method that is optimal for efficiently reacting and that can be easily realized in an automatic device. We adopted a method of injecting a substance that adsorbs to the container wall without interfering with the process. The substance to be injected is DNA that does not inhibit the reaction system.
Alternatively, polyoxyethylene sorbitan monolaurate, a nonionic surfactant, was employed.

Furthermore, in order to achieve the above object, a method was adopted in which a substance that is adsorbed on the container wall was injected into the container before reaction to cause the substance to be adsorbed on the container wall.

(Function) There is an upper limit to the number of molecules that can be adsorbed on the container wall, and the number of adsorbed substances must be sufficiently large compared to the number of DNA molecules (so that the number of molecules to be sampled on the container wall is L). Since substances other than NA are adsorbed, the amount of sample DNA adsorbed on the vessel wall can be reduced, and the reaction rate can be improved.The method of the present invention only requires the addition of an operation for injecting the adsorbent. Therefore, automation is easy.

In addition, there is a method in which a substance that will be adsorbed to the container wall is injected into the container before the reaction, and after sufficient adsorption, the container is discarded and the reaction is performed. The reaction rate can be similarly improved, and automation is easy.In addition, according to this method, if the solution is sufficiently removed to reduce the possibility of contamination, substances adsorbed on the container wall can be has the advantage that it does not pose a problem even if it has the property of inhibiting the reaction system.

〔Example〕

A first embodiment of the base sequencing process of the DNA sample preparation method according to the present invention will be described using the flowchart of FIG. In the method of this example, template DNA, template 1
) Dispensing to mix the primer, buffer, and distilled water to adjust the reaction liquid layer with the number of molecules equal to NA (operation 1)
, template 1) does not inhibit the reaction with a sequence different from NA, and the container 4
%, other species D to inject DNA to be used as adsorbent to f
NA injection (operation 2), incubation at a temperature range of 55-60°C (operation 3), cooling of the container to room temperature level (operation 4), injection of DNA polymerase (operation 5).
), aliquoting the reaction solution (step 6), injection of DNA chain elongation/terminator (step 7), incubation at a temperature range from room temperature level to 37°C (step 8), Thiase 1dJPf
1 (operation 9), incubation similar to operation 8 (operation 10), and injection of a stop agent such as formamide (operation 11).

Experimental results using this method are shown in FIG.

In this example, the primers were fluorescently identified, the DNA fragments were made to emit light using excitation light, and the emitted light was detected.6 Figure (b) shows template DNA4#Mk at 0 and 25 pm.
D when using ole and Wibin and not injecting other species DNA
This is an NA fragment spectrum, and Figure (8) shows DNA other than the template.
A is a DNA fragment spectrum obtained when the number of primer molecules is injected one order of magnitude larger than the number of template DNA molecules.

The horizontal axis of the figure is migration time, the vertical axis is fluorescence intensity, and the numbers are L) NA
This is the base length of the fragment. It is clear that the excess injected DNA reduces the adsorption amount of template 1) NA on the container wall and improves the reaction rate.

The second embodiment of the DNA sample preparation method according to the present invention is described in the second embodiment.
The basic flow shown in the flowchart in the figure is the same as in the first embodiment, but instead of step 2 of injecting DNA of another species, polyoxyethylene (20) sorbitan monomer, which is a nonionic surfactant, is used as an adsorbent to the container wall. Operation 12 of injecting laurate is adopted.

The experimental results using this method are shown in Figure 6. Figure 6 (b) shows the DNA fragment spectrum when the reaction was carried out without injecting polyoxyethylene (20) sorbitan monolaurate;
The upper part of the figure is a DNA fragment spectrum when polyoxyethylene (20) sorbitan monolaurate was injected into the annealing reaction solution to a concentration of 0.1%. Here, the primer and template DNA were supplied in equimolar amounts. Polyoxyethylene (20) sorbitan monolaurate reduced the amount of template DNA adsorbed onto the container wall, clearly improving the reaction rate.

In the first and second embodiments, the adsorbent was mixed in after preparing the reaction solution, but it goes without saying that the same effect can be obtained by mixing the adsorbent in distilled water, buffer, etc. in advance.

DNA sample i1 according to the present invention! A third embodiment of the iI manufacturing method is shown in the flowchart of FIG. Before operation 1 of dispensing and dispensing the reaction solution into a container, operation 13 of adsorbing the substance to the container wall and injecting it into the container, and operation 14 of discarding and drying the solution are performed.

PBR322 double-stranded DNA and polyoxyethylene (2o) sorbitan monolaurate were used as the adsorbent.

DNA fragment spectra with S/N equivalent to those in the upper portions of FIGS. 5 and 6 were obtained.

It goes without saying that the above method can be applied to other DNA sample reactions 1, such as limited oxygen decomposition reactions.

〔Effect of the invention〕

According to the present invention, adsorption of the DNA sample to the container wall can be prevented, and the reaction rate can be improved especially when the amount of DNA sample becomes insignificant.

[Brief explanation of drawings]

FIGS. 1, 2, and 7 are process flowcharts of the embodiment of the present invention, and FIG. 3 is a measurement diagram showing changes in I) NA fragment spectra in the base sequencing process of the conventional method.
Figure 4 is a graph that organizes the trends in Figure 3 as reaction rates.
FIGS. 5 and 6 show 41! of DNA fragment spectra of the embodiment of the present invention and the conventional example, respectively. It is an I constant diagram. Fig. No Fig. E @ type DNL Nougai Ran V type Riso A quantity mOe Tomizu

Claims (1)

[Claims] 1. A DNA sample preparation method in which an aqueous DNA sample solution and a reagent are mixed and reacted in a container, characterized by injecting a substance that does not inhibit the reaction system and adsorbs to the container wall to cause the reaction. DNA sample preparation method. 2. The DNA sample preparation method according to claim 1, characterized in that DNA that does not inhibit the reaction system is injected. 3. The DNA sample preparation method according to claim 1, characterized in that a nonionic surfactant is injected. 4. The DNA sample preparation method according to claim 3, characterized in that polyoxyethylene sorbitan monolaurate is used. 5. In the method for preparing a DNA sample according to claim 1, instead of injecting a substance that does not inhibit the reaction system and is adsorbed to the container wall, a substance that is adsorbed to the container is injected and the substance is attached to the container wall before being used in the reaction. 1. A method for preparing a DNA sample, which comprises adsorbing .