JPS63309863A - Determination of dna base sequence - Google Patents

Abstract

PURPOSE:To determine DNA base sequence in a short period of operation by bringing a reaction reagent into reaction specifically into 4 kinds of bases of labeled DNA and cleaning the nucleic acid bases in the N-glycoxide conjugated site. CONSTITUTION:The 1st regent is added to the labeled DNA and is brought into reaction specifically with 4 kinds of the bases at 20-37 deg.C in accordance with the principal of a Maxiam-Gilbert method, then the bases are subjected to the cleaning reaction in the N-glycoxide conjugated site. The 2nd reagent is in succession added to the sugar removed of the base parts to hydrolyze the phosphate bond, by which the base sequence of the DNA is determined. For example, NaIO4 is used for the G specific reaction reagent, diethyl pyrocarbonate for the G+A specific reaction reagent, iodosuccinimide for the C+T specific reaction reagent, and KMnO4, etc. for the T specific reaction reagent as the 1st reagent. Piperazine, etc., are used for the 2nd regent. Since the 1st reagent which reacts specifically with 4 kinds of the DNA bases is brought into reaction with said bases, the need for an ethanol precipitation operation is eliminated and the reaction temp. is lowered. The faster measurement is thus enabled.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to DNA base-specific cleavage reaction operation using the Maxarr+-Gi IbCrt method.

[Summary of the invention]

The present invention is based on the Maxam-Gi Ibert method.
In the A base-specific cleavage reaction, following the first reaction of removing or modifying a base in a base-specific manner, there is no need to remove reaction reagents, buffers, and salts necessary for the reaction by ethanol precipitation. The first method is safer than the conventional method, in that the second class cleavage reaction reagent is directly added to the reaction solution obtained by the first reaction, and the cleavage reaction can be carried out at a temperature lower than the conventional cleavage reaction temperature. The present invention provides reagents for modification reactions, class 2 cleavage reaction reagents, buffer solutions, and reaction conditions.

[Conventional technology]

The Maxam-Gilbert method is a DNA base sequence determination method using a chemical base-specific partial decomposition method.

In the base-specific cleavage reaction operation, first, a reaction is performed to base-specifically and partially remove or modify four types of bases, and then a reaction stop solution and ethanol are added for each base-specific reaction. The reaction reagents were removed by adding DNA, treating at low temperature, concentrating the DNA and precipitate by centrifugation, and discarding the supernatant. The method of concentrating DNA using ethanol in the presence of a salt is called an ethanol precipitation operation. This ethanol precipitation operation needs to be repeated one more time. To finish the ethanol precipitation operation, the DNA precipitate is rinsed with ethanol, and the ethanol is evaporated under reduced pressure to dry the DNA. Subsequently, a piperidine aqueous solution was added and dissolved as a second cleavage reaction reagent, and the reaction was carried out at 90°C. The above operation is shown in Figure A, Conventional Method. Until now, Maxam-Gilbe
In the rt method, a guanine methylation reaction with DMS (dimethyl sulfate) is a C-specific reaction, a depurination reaction with an acid (especially formic acid) is a G0A-specific reaction, and a hydrazitolysis of pyrimidine with hydrazine is a C1T-specific reaction. , hydrazitolysis using hydrazine in the presence of salt is recommended as a C-specific reaction, and is actually often used (Maxam A, M, G11bert W,:
Methods inenzym.

1ogy, Vol 65:Nuclic Ac1d
s, Part 1. 8cadmic PressH
1980:499-560).

=4- [Problems to be solved by the invention] According to the conventional method, after the first modification reaction, a stop solution and ethanol are required to remove reagents, and low-temperature treatment, centrifugation, and supernatant are required. The liquid removal operation and vacuum drying operation had to be performed before the second cleavage reaction, which was not only laborious and time-consuming but also economically unfavorable. In addition, the second cleavage reaction requires a high temperature of 90°C, which causes problems such as the tube inside the sunasopuki opening due to thermal expansion of the air, the reagent evaporating, and the tube flying off due to the force of the camp opening. Either a special rack was required to hold the cap down, or a reaction tube with a screw camp had to be used.

In addition, DM, which is exclusively used as the first modification reagent in the conventional method,
S and hydrazine are highly toxic and pose safety problems.

[Means for solving problems]

In order to solve the above problems, first of all, both the first class modification reaction reagent and the second class cleavage reaction reagent have low toxicity, and the second class cleavage reagent is used as the first class modification reaction reagent. It is possible to carry out the first modification reaction in an amount that does not react with the reaction reagent or does not cause a loss of potency as a class 2 cleavage reaction reagent even if it reacts, and the amount of the reagent itself and the class 2 cleavage reagent. Searching for a reagent whose reaction product with the reaction reagent is in an amount that does not adversely affect the electrophoresis performed following the base-specific cleavage reaction, and also regarding the buffer composition that provides the first modification reaction environment. In addition to consideration, we also searched for a second cleavage reaction reagent that could lower the concentration and reaction temperature.
This led to the present invention.

[Effect]

By introducing a new reaction reagent and reaction system, reaction conditions that meet the above objectives can be obtained, and there is no need to perform ethanol precipitation after the first modification reaction, and the second reaction reagent can be directly added to the second modification reaction. Can perform reactions.

At this time, by lowering the reaction temperature of 90°C, there is no need for special measures to securely fix the camp during the reaction time, making it easier to obtain stable results.

(Example) The present invention will be described in detail below. Fig. 1 shows the operational differences between the method according to the present invention and the conventional method.

In the figure, the arrow pointing toward the axis labeled DNA represents addition of reagent, the arrow pointing outward from the axis represents removal, "9" represents centrifugation, and Δ represents incubation. The operating method according to the present invention can be roughly divided into two types. Type I operation, indicated by 011) in the figure, involves adding a buffer solution and a first-class reaction reagent to the DNA, incubating it for a predetermined time, then adding a second-class reaction reagent, and incubating again for a predetermined time. Furthermore, the reaction solution is removed. On the other hand, Type 2 operation consists of adding a buffer solution, a Class 1 reaction reagent, and a Class 2 reaction reagent to the DNA almost simultaneously, then incubating for a predetermined period of time, and then removing the reaction solution. .

Type (2) reaction method can be said to be a simpler version of Type I. The table below summarizes examples of the present invention by base specificity.

The base specificities surrounded by circles in the table are combinations with the same reaction type and high reliability, and are detailed below.

G-specific reaction: 5u of standard 11 DNA [70mM Na1O
.

Add 10μl, incubate at 37°C for 15 minutes, and add 100ul of 200a+M piperazine! Add 80
After treatment at ℃ for 30 minutes, it is evaporated to dryness. Treatment at 80°C is carried out by camping. Add 40 μl of water, dissolve the sample and evaporate to dryness. The above operations of dissolving in water and drying are repeated two or more times.

G0A specific reaction: 30mM NaO to 5pl of labeled DNA
H.

After adding and mixing 10 μp of 1s MEDTA, a 3% diethylpyrocarbonate-ethanol solution is added. The following is based on the G-specific reaction.

C+T specific reaction: 100mM in 5μl of labeled DNA solution
Add 1 (Ntll) of ammonium acetate P”4.0,
Subsequently, a 20.01 mM aqueous bromosuccinimide solution is added. The following is based on the G-specific reaction.

T-specific reaction: 25+wMNa01 to 5 μl of labeled DNA
After adding and mixing 16mM EDTA, 1mM KMno
, or add 0nlOμl of NaMn. The following is G
It corresponds to a specific reaction.

Similar operations may be performed for other reactions in the list, such as adding a buffer solution or a denaturing solution to set the reaction conditions to the DNA solution, and adding the ill reaction reagent to carry out the reaction. In the case of the above-mentioned recommended reaction, only 9 kinds of solutions are required, and the pipetting operation is basically common to all 4 reactions, and the characteristic is that 10 μl each can be ordered.

〔Effect of the invention〕

With the introduction of this method, the ethanol precipitation operation is eliminated and the conventional reaction temperature of 90° C. is lowered, thereby shortening the operation time and saving labor, and eliminating the need for a low-temperature bath or centrifugal rod. Further automation is expected to simplify the equipment, significantly reduce costs, enable mass processing, and improve reliability of operation and reaction.

[Brief explanation of drawings]

FIGS. 1(a) to 1(c) are schematic diagrams showing the flow of operations to clarify the differences between the conventional method and the method according to the present invention. that's all

Claims (9)

[Claims] (1) In a base-specific cleavage reaction operation based on the principle of the Maxam-Gilbert method, four types of buffer solutions adjusted with predetermined compositions in terms of pH, ionic strength, ionic species, and chelating agent are applied to labeled DNA. It is added depending on the base, and then reacts specifically with the four types of bases at 20°C to 37°C, and can specifically cleave the nucleobase at the N-glycoside bonding site or remove it in a subsequent process. A type 1 reaction reagent that modifies the shape is added at a concentration that causes partial modification or partial decomposition, and the reaction is carried out under predetermined conditions. After that, the base moiety is removed or modified, and the base moiety is bound or modified to the sugar. A predetermined amount and a predetermined concentration of a second class reaction reagent capable of hydrolyzing the two phosphate ester bonds involved in the sugar by β-elimination reaction were added at 70°C.
DNA characterized by reacting at ~95°C for a predetermined time
Base sequencing method. (2) Hydrogen peroxide, ammonium persulfate, cidroxylamine, sodium periodate, dimethyl sulfate, propylene oxide, iodosuccinimide, bromosuccinimide, and the like as G-specific reaction reagents in the above-mentioned Class 1 reaction reagent. The DNA base sequencing method according to claim 1, which uses either thallium chloride or monoperphthalic acid. (3) The DNA base sequencing method according to claim 1, wherein the first class reaction reagent is a G+A-specific reaction reagent, which is any one of a solution, diethylpyrocarbonate, and dimethyl sulfate. (4) The DNA base sequencing method according to claim 1, wherein either hydroxylamine or hydrogen peroxide is used as the C-specific reaction reagent in the first class reaction reagent. (5) The DNA base sequencing method according to claim 1, in which the first class reaction reagent is any one of sodium permanganate, potassium permanganate, and hydrogen peroxide as a T-specific reaction reagent. . (6) DN according to claim 1, in which the first category reaction reagent is one of hydrogen peroxide, iodosuccimide, and bromosuccinimide as a C+T-specific reaction reagent.
A base sequencing method. (7) Claim 1 in which any one of piperidine, piperazine, and kinuclein is used as the reaction reagent of the second category.
DNA base sequencing method described in section. (8) The DNA base sequencing method according to claim 1, in which an alkali is used in the reaction in which it is necessary to sharply denature one DNA strand in the buffer solution. (9) The DNA base sequencing method according to claim 1, wherein the first class reaction reagent and the second class reaction reagent are added successively or simultaneously and reacted under predetermined conditions.