JPH0257978A - Method for determining arrangement of dna base - Google Patents

Abstract

PURPOSE:To simplify the determination of the arrangement of a DNA base by using the derivatives of deoxyribonucleoside-5'-trillin acid dNTP, performing DNA polymerase action, and repeating a step for returning a state into the state wherein the elongation of the dNTP derivatives can be performed. CONSTITUTION:A DNA which is to be subjected to structure analysis undergoes cloning once in colibacillus by using a vector which can form one-chain DNA such as MB phage and PVC118/119 based plasmid. The transformed one-chain DNA which is obtained by cloning is used as a template. A complementary short-chain DNA is bonded to said template DNA as a primer. Enzyme having DNA polymerase activity is made to act on the obtained template primer com posite body. Four kinds of MdNTP derivatives of dNTP having complementary labels are incorporated in the template DNA. The enzyme and the substrate are removed from the composite system of the template, primer and MdNTP. The protecting group of the newly introduced MdNTP is eliminated, and a state wherein the DNA chain can be elongated is introduced. The composite body generated at this time can enter into the next new cycle.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method and apparatus for determining DNA base sequences.

[Conventional technology]

The development of methods for determining DNA base sequences has greatly contributed to the development of molecular biology research and the success of genetic engineering. Currently, two methods are widely used, one of which is the Maxam-Gilbert method developed by Maxam and Gilbert [N. M. Maxam and W. Gilbert, Methods in Enzymology, Vol. 65, No. 499] ~
560 pages (1980)), and the other was developed by Sanger et al.
7-Le Coulson, Proceedings of the National Academy of Sciences Op. USA Vol. 74, pp. 5463-5467 (19
77)), the M13 phage improved by Metssing et al. [J. Metssing, Methods in Engineering, Vol. 101, pp. 20-78 (1983)]
) is the dideoxy method (chain terminator method).

The Maxam-Gilbert method uses the 3'- or 5'-terminal
``A specific chemical treatment is applied to each of the four types of bases in P-labeled single-stranded or double-stranded DNA to selectively cleave the DNA at each base position.The resulting degradation products are subjected to electrophoresis. Due to the difference in length of each base of DNA, [separation 1
The sequence is determined by identifying the bases corresponding to each band.

On the other hand, in the dideoxy method, a DNA fragment is cloned into M13 phage, and when complementary NA is synthesized, τ corresponding to each of the four bases in addition to dNTP is used as a substrate.
, 3'-dideoxy congeners (ddNTPs) are present. Since the elongation reaction stops at the position where each ddNTP is incorporated, the sequence can be determined in the same manner as the Maxam-Gilbert method by separating DNAs with different chain lengths by electrophoresis.

[Problem to be solved by the invention]

Each of these DNA base sequencing methods has several variations, but all are characterized by the use of electrophoresis. Therefore, reaction of DNA sample, electrophoresis,
This method requires multiple operations such as deciphering the bands on the gel obtained by electrophoresis, which is not only complicated but also unsuitable for automation. Furthermore, as the DNA strand becomes longer, the reading accuracy decreases.

[Means to solve the problem]

To summarize the present invention, the first invention of the present invention relates to a DNA base sequencing method, which is a method for determining a DNA base sequence, which is capable of being incorporated as a substrate for DNA polymerase and stopping the DNA chain elongation reaction due to the presence of a protecting group. One cycle includes the steps of performing a DNA polymerase reaction using four types of dNTP derivatives (MdNTPs) with detectable labels, then detecting the incorporated MdNTPs, and returning the MdNTPs to a state in which they can be extended. It is characterized by repeating this nine times. °The second invention of the present invention also provides a reaction tank for carrying out a DNA polymerase reaction using the MdNTP of the first invention, a reaction product separation means,
The present invention relates to a DNA base sequencing device characterized by including a means for removing a protecting group and a device for detecting a label.

The present invention will be specifically explained below.

In the present invention, DliA to be structurally analyzed is first cloned in Escherichia coli using a vector capable of producing single-stranded DNA such as M13 phage or pVc118/119-based plasmid. Next, the recombinant single-stranded DNA obtained by cloning is combined with a template and a short-stranded DNA complementary to the template DNA is used as a primer. At this time, it is also a good method to bind the template DNA and/or primer to a solid support.

Various solid phase carriers can be used.

Examples: Evacellulose, silica, polystyrene, CPG (
Control topoagrass) and their derivatives can be used, and many of these are commercially available. Binding methods can be divided into direct binding methods and indirect binding methods. As a direct binding method, for example, a template DNA labeled with biotin in advance can be used.
This is achieved by binding A to a solid support with azine and zine bonds. Commercially available reagents (such as photobiotin) are used for biotin labeling.

Avidin-conjugated carriers are also commercially available.

The indirect binding method is a method in which a primer bound to a solid phase (necessary for the initiation of DNA polymerase reaction) is bound to template DNA through hydrogen bonding. For example, the M13 universal primer used in base sequencing using the dideoxy method is a method that binds to a primer bound to a solid phase (necessary for the initiation of DNA polymerase reaction). When DNA is used as a template, a complementary strand can be formed with the template through hydrogen bonding. briar
The method of binding to a solid phase is generally known. For example, a primary amine is introduced into the 5'-end of the primer via a spacer, and this amine is attached to a crosslinker such as disuccinimidyl sperate (DSS). It can be bonded to an active carboxyl group or amine introduced into a solid phase carrier via a linker. Methods for introducing amines into primers have been established, and reagents are sold by Aglide Biosystems, for example. Furthermore, solid phase carriers into which primary amines or active carboxyl groups have been introduced are also commercially available (Pierce Co., Ltd., etc.).

As another indirect method, for example, N
A can be bound to a solid phase, and template DNA can be bound to this by hydrogen bonding. In this case, since the DNA to be bound to the solid phase does not function as a primer, it is desirable to bind the 3'-end to the solid phase.

Such methods are known, and for example, a method that does not cause cleavage between the solid phase and the DNA during the deprotection process with ammonia when chemically synthesizing the necessary sequence may be employed.

i.e. 1 at both ends, such as 1,5-pentane-diol.
After one of the diols having a class hydroxyl group is protected with a dimethoxytrityl group (DMTr), the other is phosphorylated and then bonded to a functional group on a solid phase support. The solid phase carrier thus obtained can be used for chemical synthesis of DNA after removing DMTr. The synthesized DNA is deprotected with ammonia to remove all protecting groups on the DNA, but the 37-end of the DNA remains bound to a solid support.

Through the above process, a complex of template DNA and primer that can be bound to a solid phase is obtained. This mold/
Enzymes with DNA polymerase activity (DNA polymerase 11 Klenow fragment,
Taq polymerase, reverse transcriptase, etc.) is activated to incorporate complementary MdNTPs into the template DNA, thereby stopping DNA chain elongation. Next, the enzyme and substrate are removed from the system containing the template/primer/MdNTP complex. Furthermore, the protective group (and label) of the newly introduced MdNTP is removed, leading to a state in which DNA chain elongation is possible. The complexes generated here can enter the next new cycle.

In addition, in the MdNTP uptake reaction, incomplete or N
In order to remove noise caused by the A chain, four types of ddNTPs can be added to the reaction system every several tens of cycles.

Identification of incorporated MdNTPs is performed by measuring the label. As the label, dyes, fluorescent dyes, ultraviolet absorption substances, luminescent substances, radioactive isotopes, pyrophosphoric acid released from MdNTPs, and the like can be used. The sign is 4
Depending on the type of base, a specific label (different wavelength, nuclide, etc.) can be attached, or the same label can be used. When using the same label, identification can be performed by comparison with an unlabeled substance using reaction vessels compatible with four types of bases. The label may be measured after being detached from the MdNTP, or may be measured in the form of a template/brimer-MdNTP complex. When the protecting group is labeled, deprotection and removal of the label can be performed simultaneously. Labels are detected depending on the labeling method. For example, dyes can be detected using a visible partial photometer, fluorescent dyes can be detected using a fluorometer, ultraviolet absorbing substances can be detected using an ultraviolet partial photometer, luminescent substances can be detected using a luminometer, and radioisotopes can be detected using a scintillation counter. Alternatively, each label may be examined by liquid chromatography, separated based on differences in retention time, and then detected using the above-mentioned equipment.

When using dyes, fluorescent dyes, or ultraviolet absorbing substances as labels, it is desirable that these have high extinction coefficients or fluorescence yields. The fluorescent label can be selected from commercially available labeling agents for fluorescence detection, such as fluorescein isothia-4-) (FITC), tetramethylrhodamine isothiabase (TRITC), β-D-
Galactosylumbelliferon, Texas Red, N
BD Recl ReF (7-chloro-4-nitrobenz-2
-oxa-1,3-diazole).

MdNTP is not particularly limited as long as it meets the above purpose. However, to stop the DNA chain elongation reaction, 3
It is desirable that a protecting group is bonded to the 1 position, and the protecting group is preferably one that can quickly and completely regenerate the free 3'-'OR group. For removing the protecting group, there are biochemical methods using enzymatic reactions, chemical methods using specific reagents, pH changes, etc., and physical methods using temperature changes, electromagnetic fluids, etc. Examples of enzymes used in the deprotection reaction include non-specific carboxyesterase (EC, 3,1
, 1, 1) and 0-glucosidase [EC, 3, 2,
1) etc. The enzyme used depends on the type of MdNTP and enzyme used, but the enzyme and the binding mode are not particularly limited. As the 3'-protecting group to be removed by chemical or physical methods, known hydroxyl-protecting groups and protecting groups modified by attaching labels to them can be used. 3′ is eliminated under relatively mild conditions.
- As a protecting group, trityl [elimination conditions = 80% acetic acid, 1
00°C, 10 minutes], trimethoxytrityl [80% acetic acid-dioxane, room temperature, several minutes], pivaloyl (I N N
aOH, 0°C, 30 minutes], trifluoroacetyl (p)
111, room temperature, immediately], dihydrocinnamoyl [chymotrypsin], p-nitrophenoxycarbonyl [imidazole, room temperature, 30 minutes], isobutyloxycarbonyl (0,4N NaOH 1 room temperature, 30 minutes), etc.Protecting groups When attaching a label to a protective group, a radioactive element can be used, or a label can be added to a part of the protecting group.
They can also be bonded via a mino group, an amide bond, a sulfonamide bond, a thiourea bond, a carbon-carbon bond, or the like. It is also a good method to insert a spacer of any chain length between the 3'1 site and the label. The presence of this spacer allows the enzymatic reaction to occur smoothly. The chain length of the spacer is not particularly limited, but as shown in (1), n = 1 to 20 is preferred, and n = 4 to 12 is particularly preferred.

The preparation of MdNTPs was performed using the N
, N'-carbonyldiimidazole (CDI) method [B.B. Goteyk, N.K. Krajewski, N.B. Tarsova, B.B. Brigin,
Tetrahedron, No. 26
Vol., pp. 4419-4433 (1970)) is convenient. This method involves activating the carboxyl group of the compound to be introduced with CDI, and then converting it into a dNTP.
react with the 3'-hydroxyl group of The introduced protecting group does not react with the base moiety at all, but reacts with the 3'-hydroxyl group, and the reaction conditions are very mild, so dATP JpdG
The target MdNTP can be obtained without damaging r-phosphate, which is easily decomposed such as TP. Fluorescently labeled MdNTP (II) was prepared according to the method of Schieffer et al. [G. Schieffer, G. Onur, European Journal of Biochemistry Vol. 97, pp. 415-424 (1979)]. It can be synthesized by Further, (to) is synthesized according to the method of Hiratsuka [T. Hiratsuka, Biochimica Kogyo, Biophysicica Ken Acta, Vol. 742, pp. 496-508 (1983)]. As an alternative method, the desired MdNTP can also be obtained by triphosphorylating the 5'-position of a dNTP modified with a 3'-hydroxyl group by a known method.

(I) (■) (III) In the formula, B is a nitrogen base such as adenine, guanine, cytosine, or thymine, Ind is a label, and X is N)1. ．． 01
S, carbonyl, thiocarbonyl, or methylene,
Further, n represents an integer from 1 to 20.

The present invention also provides a DNA base sequencing device that automates the DNA base sequencing method described above.

That is, it includes a reaction tank for carrying out the above-mentioned DNA polymerase reaction, a washing solution tank for isolating reaction products, a reagent tank for removing protecting groups (and labels), and a label detection device.

Using this device, the following reaction system can be set up.

MdNTP is incorporated into a DNA strand as a substrate for DNA polymerase, and elongation of the DNA strand is stopped when one base is elongated. To restart elongation of the DNA strand, the protecting group of the DNA strand (preferably the 3'-terminus) whose elongation has been stopped is removed. MdN incorporated either before or after deprotection
Identify the label of TP. The DNA base sequence is read using a reaction system in which the steps up to this point constitute one cycle. More specifically, D
The NA polymerase reaction tank contains a template bound to a solid support.
Filled with brimer complex, MdNTP of formula (I) and D
After flowing NA polymerase from its storage tank to extend the chain length by one base, the reaction tank is washed with a buffer solution in the wash solution tank, and then esterase is flowed through the protection group removal reagent tank to remove the labeled protection. For example, one cycle is completed by separating the group, detecting it with a label detection device, and washing the reaction tank again.

〔Example〕

The present invention will be explained below using specific examples, but the present invention is not limited to these specific examples.

Solid phase bound primer 0.2 nmole, M 13m
pl 85g DNA 0.2 nmole and 10x concentrated polymerase buffer (70mM) 'JS Hydrochloric acid pH 7.5
, 1mM EDTA, 20mM Na
0.44 (70mM MgC) was mixed to make the total amount 14. After being held at 60°C for 20 minutes, it was allowed to cool to room temperature. (The above operation is called template-primer preparation.) On the other hand, 3'-acetyl dNTP (AcdNTP) protected with four types of acetyl groups was prepared using Marian's method [M-Marian, Microchemical Fist Journal, Vol. 29, No. 219 ~227 pages (1984)),
Only one type of acetyl group was labeled at °C and MdN
A mixed solution of TP (1 nanomol/d aqueous solution each) was prepared as follows.

Mixture A: MdATP%AcdGTP%AcdCTP
, AcdTTPG mixture: AcdATP, Md
GTP, AcdCTP, AcdTTPC mixture:
AcdATP, AedGTP%MdCTP, A
cdTTPT mixture: AcdATP, AcdGT
Take 0.5 rfLt of P, AcdCTP%MdTTPA, G, C, and T mixtures into separate tubes, and add 0.2 d of the previously prepared template-primer mixture.
was added to each tube, and 50 units (0.3 mj) of reverse transcriptase was added to each tube, followed by shaking at 20°C for 10 minutes. After separating the template-brimer from each reaction solution and washing free ACdNTPs and MdNTPs, 0.2 mm of a buffer containing 1 unit of purified pig liver-derived esterase was added, and the mixture was shaken at 20°C for 5 minutes. After separating the template and primer, the label released from each tube was analyzed using a liquid scintillation counter. As a result, radioactivity was observed only in the tube containing the G mixture, and Md
It was found that GTP was incorporated. Then, reverse transcriptase and each mixed solution were newly added to the 3'-OH released in each tube to perform a polymerase reaction, and then a cycle of performing an esterase reaction was repeated. As a result of repeating this cycle 10 times, the MdNTPs incorporated in each cycle were as follows.

Cycle 4 1 2 3 4 5 6 7 8 9
10MdNTP G T T G T
The sequence at A A A A is M13 phage 1
It was completely complementary to the sequence upstream of the primer binding site of the double-stranded DNA.

〔Effect of the invention〕

As explained in detail above, the present invention has made it possible to determine DNA base sequences without using complicated electrophoresis.

Claims (1)

[Claims] 1. DNA that is incorporated as a substrate for DNA polymerase
A step of carrying out a DNA polymerase reaction using derivatives of four types of deoxyribonucleoside-5'-triphosphates (dNTPs) that can stop the A-chain elongation reaction by the presence of a protecting group and that have a detectable label; a step of detecting the incorporated dNTP derivative; and a step of detecting the incorporated dNTP derivative;
A method for determining a DNA base sequence, characterized in that the step of returning a derivative to a state capable of elongation is one cycle, and the cycle is repeated. dNT modified with a protecting group with a label at the 2,3'-position
2. The DNA base sequencing method according to claim 1, which uses a P derivative. 3. dNTPs labeled with dyes, fluorescent dyes, ultraviolet absorption substances, luminescent substances or radioactive isotopes as detection means
2. The DNA base sequencing method according to claim 1, which uses a derivative. 4. The DNA base sequencing method according to claim 1, which comprises performing a DNA polymerase reaction with the primer and/or template DNA bound to a solid phase carrier. 5. DNA base sequencing characterized by comprising a reaction tank for carrying out a DNA polymerase reaction using the dNTP derivative according to claim 1, a reaction product separation means, a protecting group removal means, and a label detection device. Device.