JPS63241356A - Determination of base sequence for polynucleotide and/or oligonucleotide and analyzer using the same - Google Patents

Abstract

PURPOSE:To simplify operation along with a higher accuracy, by causing simultaneous reaction between a sample, a primer complementary thereto, non-labeled four kinds of nucleotide triphosphate and differentially labelled four kinds of modified nucleotide derivatives. CONSTITUTION:Polynucleotide or oligonucleotide desired to determine sequence, a single-chain nucleotide sequence (primer) complementary to a sequence to be arranged as base sequence, non-labelled four kinds of nucleotide triphosphate adenine, guanine, cytosine and thymine (uracil), differently labeled one or more kinds of modified nucleotide derivatives of adenine, guanine, cytosine and thymine (or uracil) are held into one reaction container to make hybrid forming conditions. This enables simultaneous labeling of one or more kinds only with single reaction to determine sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for determining the base sequence of polynucleotides and/or oligonucleotides, and an analysis device used therefor. More specifically, the present invention relates to a labeling compound used for easily and accurately detecting an analyte in polynucleotide and/or oligonucleotide sequence analysis, particularly DNA base sequence analysis.

The development of reliable base sequencing methods for DNA (deoxyribonucleic acid) and RHw) (ribonucleic acid) is based on recombinant DNA
It has become one of the keys to the success of technology and genetic engineering. As a base sequencing method, the Sanger method (Proc
, Natl, Acad, Scl USA, 5
463 (1977), Methods in En.
z, , , Hexagram 5a-580 (198Q)), # and the Maxam-Gilbert method (Methods in Enz,
, 65+499-559 (1980), two different methods are widely used.

The method developed by Sanger et al. is a base sequencing method that utilizes the polymerase reaction of DNA polymerase, and is also called the dideoxy (chain end) method, and is the method most commonly used in recent years. In 1969, Atkinson et al.
Atkinson, K. R. reported that NTP (guideoxyribonucleotide) acts as an inhibitor of DNA synthesis by DNA polymerase I.
, et al, , Biochemi 5try,
8.4897-4904 (1969)), then Sa
Based on this fact, Nger et al. proposed a method for terminating the polymerization reaction of DNA polymerase using 4 liters of ddNTP.
presented a method for sequence analysis (Sanger, F.,
et al, proc, Natl, Acad, S
ci, USA, 74.5463 (1977)).

In this method, the target DNA fragment is, for example, M
Cloned into a single gJDNA frame such as 2S,
These 77-di DNAs are used as templates for primed synthesis of complementary strand DNA by the Klenow fragment of DNA polymerase I. ζThe primers required for this primed synthesis of complementary strand DNA are synthetic oligonucleotides or restriction enzyme-digested fragments that are specific to the region of the M13 vector near the 3' end of the cloned insert. isolated from the original DNA to which it hybridizes. One M2S containing the target DNA fragment ff1
This primer is annealed to the DNA, and a complementary 1 mRNA synthesis reaction is performed using the Klenow fragment of DNA polymerase I. At this time, a 481 type sequencing reaction system was created, and each reaction system contained four types of dNTPs as substrates.
(deoxyribonucleotide) and one type of ddNTP as a chain terminator are allowed to coexist. For example, when ddGTP is added, when dNTPs are sequentially added and complementary*DNA is extended in the 5l-37 direction, dGT
For each addition reaction of P molecules, some complementary strand DNA is generated with a certain probability.
The fragment incorporates ddGTP. In this case, since ddGTP lacks the ζ hydroxyl group at the 3' position of the sugar moiety, the elongation reaction stops there and is selectively terminated. Therefore,
ddGTP 1 ddCTP in the reaction system of 4al species, respectively.
By adding . The relative concentration ratios of dideoi and deoxy forms added to the reaction system are adjusted so as to produce terminations that correspond to all possible chain lengths that can be separated by gel electrophoresis.

If the alpha position of one dNTP is labeled with a radioactive isotope such as 32p during the above priming synthesis reaction,
After separating the DNA fragments obtained from each of the four types of synthesis reactions by polyacrylamide gel electrophoresis, the separated DNA
The base sequence can be determined by analyzing the pattern of the A fragment using an operation such as autoradiography and examining the four band patterns obtained as a result.

On the other hand, the method developed by Maxam and Gilbert determines base sequences by chemically cleaving specific bases, and is also called the chemical decomposition method.

In this method, single-stranded or double-stranded DNA labeled with a radioactive isotope such as 32p at either the 3'-end or the 5'-end is subjected to four types of base-specific chemical decomposition reactions. Partially disassemble using . That is,
Selective chemical cleavage occurs on one or two of the four types of nucleotide bases. The conditions for the cleavage reaction are adjusted so that most of the end-labeled purified DNA fragments can be separated by polyacrylamide gel electrophoresis. By separating the obtained partial degradation products by polyacrylamide gel electrophoresis and analyzing the band pattern by performing autoradiography, etc.
The base sequence can be determined in the same manner as the Sanger method described above.

All of these base sequencing methods are widely used and each has several variations, but they have the following serious drawbacks.

First, in the conventional method, as mentioned above, the DN
It was necessary to use a radioisotope as a label to determine the position of the A band. Although labeling with radioactive isotopes is excellent in sensitivity, it has the following drawbacks when used.

1) Skill is required for handling.

2) It is subject to strict legal regulations, requires special equipment and measuring equipment, and can only be handled in limited locations.

3) You have a health problem.

4) There is a problem with disposal after use.

5) Since pre-orders are made in conjunction with the half-life period, the experimental date is limited by this.

Second, conventional DNA sequencing methods 1 require a single radiolabel, namely 3H,+4 (, 32p, 3
In order to identify all bands on a gel using either 581 or 1251, it was necessary to charge each group of DNA fragments generated in four types of synthesis reactions with a separate gel lane. . Band mobilities between four different lanes must therefore be compared, with the added problem that, due to the nature of radiographic techniques, the autoradiographic band image is broader than the band itself. , limiting the resolution of the observed bands and limiting the size of sequences that could be read from the gel.

Moreover, the irregularity of band mobility from lane to lane in the gel makes automatic reading of autoradiography difficult;
Therefore, these readings are better with the naked eye than with a computer; this reading operation is very time consuming and requires skill.

Therefore, as an alternative to the conventional base sequencing method using radioisotopes, which has the various drawbacks mentioned above, this method does not require special equipment or equipment, and is simple, biologically safe, and highly accurate. Its appearance is awaited, and its development is progressing.

For example, according to Japanese Patent Application Laid-open No. 60-22086, DN
In base sequencing method A, by binding four different labeling compounds to the primers used in the Sanger method (dideoxy method), acrylamide gel electrophoresis for sequence analysis, which conventionally required four lanes, can be performed.
It became possible to perform analysis using only runes, and therefore automation of the analyzer became possible. That is, ply 7-, each of which has been labeled in four different ways, is combined with lfi, a dideoxyribonucleotide, and used to perform a priming synthesis reaction with the Klenow fragment of DNA polymerase I, and then these four types of reactants are combined. A method for determining the DNA sequence by mixing the mixture and charging it to a polyacrylamide gel rune has been disclosed. Here, since a fluorescent substance or a chromogenic substance is used as a label instead of a conventional radioactive isotope, it is possible to label a plurality of N classes. As a labeling method, when synthesizing the primer, one extra 5'-aminothymidine having a primary amino group is added to the 5'-side of the conventional primer.
After the primer is synthesized, a labeled primer is obtained by binding a fluorescent substance or a chromogenic substance to the primary amino group at the 5' position. Next, using this labeled primer, the base sequence is determined by the dideoxy method. It is true that this method has made it possible to greatly improve the automation of sequence analysis, but it is difficult to generate approximately the same level of reaction in the four types of priming synthesis reactions, and as a result, the four types of labeled The variation in the strength of the gradation becomes large, often resulting in errors, and the problems of the conventional 8Mi, especially the vI degree, have not been solved to a great extent.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to provide a polygon system that is significantly easier to operate than the conventional method and has improved accuracy. Nucleotides and/or
Another object of the present invention is to provide a method for determining the base sequence of oligonucleotides.

Another object of the present invention is to provide a method for chemically synthesizing labeled dideoxynucleotide derivatives for easily and highly accurately determining the base sequence of polynucleotides and/or oligonucleotides.

Yet another object of the present invention is to provide polynucleotides and/or
Another object of the present invention is to provide a new analytical device (system) for easily (and highly accurately) determining the base sequence of oligonucleotides.

(Means for Solving the Problems) In order to achieve the above object, the method for determining the base sequence of polynucleotides and/or oligonucleotides by the chain termination method of the present invention provides a method for determining the base sequence of polynucleotides and/or oligonucleotides under hybridization conditions. or an oligonucleotide, a single-stranded nucleotide sequence (primer) complementary to the sequence to be sequenced, an unlabeled adenine, guanine, cytosine, and thymine (or uracil) nucleotide triphosphate; Four types of modified nucleotide derivatives, adenine, guanine, cytosine, and thymine (or uracil), which are labeled with more than one type, are added together into a single reaction vessel, thereby allowing one type of modified nucleotide derivative in a single sequencing reaction. It is characterized in that the above labeling is performed simultaneously.

The method for determining the base sequence of polynucleotides and/or oligonucleotides using the chain termination method of the present invention uses four types of dideoxynucleotide derivatives in which each of adenine, guanine, cytosine, and thymine (or uracil) is labeled with a different SW4 substance. This method is characterized in that different m of the four types can be simultaneously, independently and selectively identified in a single sequencing reaction using .

The method for determining the base sequence of polynucleotides and/or oligonucleotides by the chemical decomposition method of the present invention comprises
It is characterized in that the '-end is labeled with a labeled dideoxynucleotide derivative and then subjected to a sequencing reaction.

The analytical device for determining the base sequence of polynucleotides and/or oligonucleotides of the present invention includes colored, fluorescent, chromogenic, or ligand fB-labeled polynucleotide and/or oligonucleotide raw materials, electrophoresis gels, etc. a zone containing the sl-labeled polynucleotide and/or oligonucleotide, means for introducing the sl-labeled polynucleotide and/or oligonucleotide into the zone; optical measurement means for monitoring.

The analytical device for determining the base sequence of polynucleotides and/or oligonucleotides of the present invention is an analytical device for determining the base sequence of polynucleotides and/or oligonucleotides, and comprises:
Fluorescent, chromogenic or
and optical measurement means to monitor the gel as it moves through the gel.

The present invention will be explained in detail below.

Labeling in the Sanger Method In the method disclosed in JP-A-60-220860, a mixture of reactants obtained in four types of priming synthesis reactions is combined with a primer by different labeling compounds of the 4'y4 class. When performing electrophoretic analysis, it was possible to perform it using a run. However, it is difficult to perform the four types of priming synthesis reactions to the same degree, and therefore the variations in the intensity of the four types of labeling become large, which tends to cause errors in reading, and problems with accuracy remain.

Therefore, in the method of the present invention, in order to perform four types of priming synthesis reactions at once and perform each reaction equally,
Gideoxyribonucleotide (a reagent to stop the priming synthesis reaction)
ddNTP) is characterized by introducing a label into the IC. That is, four types of guideoxyribonucleotides ddATP% ddGTP, ddcTP and dd
By introducing different labels to TTP (or ddUTP), 18Ill types of primers and 4 types of monomers (dATP, dGTP, dCTP%
dTTP (or dUTP) or ATP%GT
P.

CTP%TTP (or UTP)) and each of these differently labeled ddATP, ddGTP
% ddcTP and ddTTP (or ddUTP
) into one reaction vessel, add adenine, guanine, cytosine (0, and thymine (
or uracil υ), it has become possible to simultaneously attach independent and selectively different labels. As described above, according to the method of the present invention, four types of priming synthesis reactions can be performed at once in one reaction vessel, so the reaction conditions for A, G, C, and T (or U) are all ( [can be the same and therefore complete] with the same intensity, four types of 8
It is possible to make a single identification. Therefore, the method not only simplifies the conventional method for determining base sequences, which requires time and effort, but also greatly improves the accuracy of analysis, and is a method suitable for automation of base sequence determination.

Labeling method of the present invention using Maxam-Gilbert method I
According to the above, labeling of polynucleotides and/or oligonucleotides whose base sequences are to be determined by the Maxam-Gilbert method is carried out by using the labeled guideoxyribonucleotide derivative of the present invention using terminal deoxyscleotidyl transferase (TO,T). This is done by selectively adding only one nucleotide to the 3'-end of the polynucleotide and/or oligonucleotide in question. Next, regarding the differently labeled polynucleotides and/or oligonucleotides,
One or four base-specific chemical reactions are performed and then filtered into a polyacrylamide gel using conventional methods or, in the latter case, by combining the 4m reactants together.
The sequence can be analyzed by charging the tube and performing electrophoresis. However, in this method, two types of labels exist at the same time in one band in the G%A-specific reaction lane and the C,T-specific reaction lane, so each of them can be analyzed separately. An analytical device is required, and it may require some ingenuity in terms of equipment compared to the Sanger method.

Chemical Synthesis The labeled guideeoxyribonucleotide derivative used in the method for determining the base sequence of polynucleotides and/or oligonucleotides of the present invention is shown in the following structure.

Here, B represents a pyrimidine derivative or a 7-deazapurine derivative which is a nucleobase covalently bonded to the C-1' position of the sugar moiety. If B is the former, the sugar is the nucleic acid salt i
is attached to the N-1 position of the nucleobase, while in the latter case the sugar is attached to the N-9 position of the nucleobase.

Sig is a reporter group for detection and is a chemical group containing at least one carbon atom. R is reporter
This is a spacer group that binds group Sig and nucleobase B. When B is a pyrimidine conductor, R is bonded to the 5-position of the pyrimidine derivative, and when B is a 7-deazapurine derivative, R is bonded to the 7-deazapurine derivative.
It is connected to the position. X represents mono-, di-, or triphosphoric acid.

Y and 2 are hydrogen.

The labeled dideoxynucleotide derivative of the present invention shown in the above structure must have the following properties.

1) It can serve as a substrate for enzymes such as polymerase, terminal deoxynucleotidyl transferase, and reverse transcriptase. 02) The 5' hydroxyl group is a phosphate ester.

3) The 3' end must be a deoxy form to stop the reaction.

4) The polymerase reaction can be stopped without error at adenine, guanine, cytosine, or thymine (or uracil).

5) The labeling substance (reporter group Sig) and spacer group R may interfere with hybrid formation or
Alternatively, reactions by enzymes such as polymerase, terminal deoxynucleotidyl transferase, and reverse transcriptase must not be interfered with.

The reporter group Sig for detection is thus attached to a sterically permissive site of the nucleotide unit to be modified thereby. Here, the sterically permissive site of a nucleotide unit is a position on the nucleobase of the nucleotide unit, where the nucleotide unit is modified by binding a substituent to that position. , the hybridization of the product oligonucleotide to the complementary nucleic acid component is not significantly hindered, and the presence of the substituent is effective for polymerase, terminal deoxynucleotidyl transferase and reverse transcribease of the nucleotide. It means a position and structure that does not inhibit its function as a substrate for enzymes such as. Such sterically tolerant sites are the C-7 position of 7-deazapurine derivatives and the C-5 position of pyrimidine derivatives.

On the other hand, sites that should not be modified include NH2 at the N-1 and 6-positions of adenine bases, and N-1 of guanine bases.
, NH2 at position 2 and O-6 position, 01N-3 at position 2 of cytosine base and NH2 at position 4 and N- of thymine base.
Examples include O at the 3rd and 4th positions. In general, substitutions with heteroatoms (N or 0) should be avoided. When a substituent is introduced at the C-8 position of a t-purine derivative, the base and sugar take a Syn-type coordination position, which reduces the ability to form a hybrid when the double helix is cloth-wound (normal or NA). and is generally undesirable. When purine derivatives are further introduced into the N-7 position, the nitrogen becomes quaternary, and the glycosidic bond between the sugar and the base moiety is easily broken, resulting in a so-called depurination reaction (Japanese Patent Laid-Open No. 61-5759).
Publication No. 5).

The substituent R as a spacer group for modifying a nucleotide unit with a reporter group Sig can function as one or more reporter groups or can be attached to one or more reporter groups. It has the following characteristics. In the present invention, the substituent R bonded to the reporter group S1g can generally be said to exhibit nucleophilic properties. Examples of such substituents include primary amino groups, aromatic amino groups, thiol groups, carboxyl groups, and hydroxyl groups. In substituted pyrimidine or 7-deazapurine bases, the substituent R contains one or more carbon atoms. From the above, it is preferable that the substituent (spacer group) R takes the form of a carbon group represented by the following chemical formula.

-C1ICR, R2, -ZCHR, R.

Here, R1 is hydrogen or a lower alkyl group of c, 6, R2 is cnH2ny, and 2 is a polyvalent heteroatom. Here, n is θ~20. Y is hydrogen or at least one amide group, substituted amide group, nitrophenyl group, substituted nitrophenyl group, substituted amino group, ester group, substituted carboxyphenyl group, substituted aminoalkylphenyl group, or substituted aminobenzenesulfonic acid group. be.

The modified pyrimidine base in the present invention is characterized by its C
The -5 position is substituted, and representative examples are uracil and cytosine base derivatives shown by the following structural formulas.

The 7-deazapurine base modified in the present invention is substituted at the C-7 position, and typical examples are 7-diazaadenine and 7-diazaguanine base derivatives shown by the following structural formulas.

For reporter groups SiH, at least one reporter group is a functional colored, fluorescent, luminescent or ligand-recognizing reporter group.

Examples include fluorescein, rhodamine, acridinium salts, nitrophenyl, dinitrophenyl, benzenesulfonyl, vitamins, luminol, isoluminol, luciferin, dioxetane, dioxamide or biotin, and substitutes or adducts thereof.

Representative examples of fluorescent labeling substances include fluorescein inthiocyanate (FITC) and eosin isothiocyanate (
E I TC), Tetramethylrhodamine thiocyanate (TMRITC), Substituted rhodamine isothiocyanate (XRI TC), 7-Fluoro-4-nitrobenzo-2-ox” P it 3-diazole (NBDF)
Examples include. Fluorescent labels are, for example, FITC for ddATP.

EITC is used for ddCTP, TMRITC is used for ddGTPlζ, and XRITC is used for ddTTP.

The reporter group Sig is bound to the dideoxy compound via a spacer group R.

In addition, a chelating group such as ethylenediaminetetraacetic acid group was bonded to the dideoxy compound using the reactive amino group of spacer group R, and a substance with long-lived fluorescence such as europium was bonded to it. using things,
By measuring delayed or long-lived fluorescence,
Significantly reduces background and signal-to-noise ratio (
It is also possible to perform good measurements of S/N ratio). If desired, the reporter moiety can include bound or attached magnetic moieties, such as magnetic oxides or magnetic iron oxides, so that such polynucleotides can be detected by magnetic methods. . Furthermore, it is also possible to bind a substance such as biotin and then use a detection system with high detection sensitivity such as a biotin-avidin system. In any case, the method of the present invention does not require radiolabeled nucleotide triphosphates in the sequencing reaction.

The labeled dideoxynucleotide derivative of the present invention as described above is produced by the following method.

1) A compound having the following structure, where Y=OH and Z=O
H1 or Y=OH or Z=H, or Y=H or Z=
First, a compound that is OH is converted into a dideoxy compound with Y=Z=H.

2) The dideoxy compound is reacted with a mercury salt in a suitable solvent to produce a mercury compound having the following structure.

3) The mercury compound and the chemical group represented by R are reacted in the presence of a palladium catalyst to produce a compound having the following structure. Here, N is a reactive terminal functional group or Si
It is g.

4) If N is not Sig, convert the chemical to Sig
A compound having the following structure is synthesized by reacting with

To combine the portions of the sequencing reaction, the mixture of four reactants is loaded into the 5% polyacrylamide gel column shown in FIG. 5 from its upper reservoir. A high voltage is applied to the column to cause electrophoresis of the labeled polynucleotide and/or oligonucleotide fragments into the gel. Label molecules that differ in length by a single nucleotide are electrophoretically separated in this gel matrix. At or near the bottom of the gel column, bands of labeled molecules are dissociated from each other and passed through a detector.

The detector detects colored, fluorescent or colored bands of labeled molecules in the gel to determine their color and detect their corresponding nucleotides. This method provides polynucleotide and/or oligonucleotide base sequences.

Detection device There are many different detection methods for detecting labeled molecules separated by molecular weight (length) by polyacrylamide gel electrophoresis, but basically the method described in JP-A-60-220860 The apparatus described in the publication can be used. Furthermore, existing equipment or improved products thereof can be used to detect separated labeled molecules using HPLC. Typical detection methods are shown below.

1) Detection of fluorescence excited by different wavelengths of light for different dyes.

2) Detection of fluorescence excited by light having the same wavelength for different dyes.

3) Elution of molecules from the gel and detection by chemiluminescence.

4) Detection by absorption of light by molecules.

5) Magnetic detection by magnetism.

6) Detection with affinity substance.

The methods for detection are not limited to those exemplified here.
It is obvious that various methods are included.

Examples of fluorescent materials have already been described, but they may also be combined with chemiluminescence-exciting agents, such as dioxyethanedione, and flowed directly into a detector chamber where the emitted light can be measured. In this case, no excitation light source is required and
The background signal is much lower than in fluorescence, resulting in a higher signal-to-noise ratio (S/N) and improved sensitivity. Furthermore, although the absorbance method is simple in terms of equipment, it has the drawback of inherently low sensitivity. The method using aph(nity) substances is relatively simple as a detection method.

As the optical measurement means of the detector used in the various detection methods described above, a spectrophotometer, a fluorometer, an optical luminescence detector, or the like is suitable.

The above detection device is placed in front of the computer. At each inspection,
The computer receives signals proportional to the current measurement signal strength for each of the four types of colored, fluorescent or chromogenic signals. This information indicates which nucleotide will now terminate a polynucleotide and/or oligonucleotide fragment of a particular length in the observation window. The sequence of colored, fluorescent or colored bands at this point gives the base sequence.

EXAMPLES The examples described below are illustrative of various aspects of the invention, but are not intended to limit the scope of the claims in any way.

Fluorescent or piotin-labeled 2' according to Figure 1.
, 3'-dideoxy-5-(3-1mino)-allylcridine-5'-triphos heptaate.

10 rnmol of 21-dioxyclidine
0me of anhydrous pyridine, 15 mmol of dimethoxytrityl chloride (DMTr-CI) was added, and the mixture was stirred at room temperature for 18 hours. 50% solution
After dilution with 0 tt' of chloroform, the oil was oiled with αIN sodium bicarbonate. The chloroform phase was dried and evaporated to dryness under reduced pressure. 2'-deoxy-51-0-dimethoxytritylcrisine was obtained using silica glu column chromatography using a chloroform-methanol step gradient. The yield was 83%.

The synthesis of the guideeoxy compound shown below from (1-2>IA to (1-5)) was performed using the method of Horwitz et al. (J, P
, Horwitz, et al., Nucleosid
es, nucleotides, 27, 3045 (
1962) ) lζ followed.

Synthesis: 2'-deoxy-5' obtained in section (1-1) of this example
9.5 mmol of methanesulfonyl chloride (MsCl) was slowly added dropwise to a solution of 7 mmol of -0-dimethoxytritylcridine in pyridine under water cooling. After reacting for 24 hours under water cooling, 11 nl of water was added,
The reaction was stopped. This was slowly added dropwise to the solution in 800 m ice water, and the resulting precipitate was filtered, washed with water, and air-dried. The pale yellow product was dissolved in a small amount of amethane and slowly dropped into 1 ton of ice water again, and the colorless precipitate was filtered and collected. The yield was 85%.

Synthesis: 21-deoxy-31-0-mesyl-5'-〇-dimethoxytritylcridine L2mm obtained in section (1-2)
ollζ, α24M Calic A-tert-putoxite (KO”Bu) in dimethylsulf7oxide solution 10
After adding - and reacting at room temperature for 1 hour, the mixture was added dropwise to 250 - of ice water. After neutralization with dilute acetic acid, the precipitate was centrifuged, washed with water, and air-dried. This was dissolved in 151 nl of ethanol, 80- of benzene was added, and concentrated under reduced pressure.
Water was completely removed azeotropically. A small amount of benzene was added, and the mixture was again concentrated under reduced pressure, and the resulting precipitate was recrystallized from ethanol. The yield was 80%.

Synthesis of Nene: 1 mm of 5'-0-dimethoxytrityl-zl,al-guideoxy-2'-clidinene obtained in section (1-3)
To 20 me of a chloroform solution of 100 ml, under water cooling, Ll equivalent of dry hydrogen chloride gas was slowly added with stirring, and about 30 me
Detritylation was carried out for 1 minute under water cooling. The generated precipitate was filtered, washed with chloroform, and then recrystallized from benzene-ethanol (90:10 v/v). The yield was 60%.

Composition: zl, 31-guideoxy-2 obtained in section (1-4)
'-Clidinene (L5 mmol dioxane solution 25 d
To this, 115 μm of dicumu carbon catalyst (10%) was added, and the mixture was reduced at 25° C. for 1 hour under 1 atmosphere of hydrogen pressure. After the reaction, the catalyst was filtered off, and the filtrate was dried under reduced pressure. The residue was recrystallized from benzene-petroleum ether. The yield was 80%.

The triphosphorylation shown below as (1-6) and (1-7) IJII was performed using the method of Ru5sell et al. (J, Org, Che.
m.

80, 4889 (1969)).

1 mmol of 2',3'-guideoxyclidine obtained in section (1-5) was
) In solution 201rI, 4 mmol of triimidazole 7-osulfur oxide and 1001
1,1'-carbonylciimiguzole (n9) was added, and the mixture was stirred overnight at room temperature to react. Next, 2-triethylamine and 5 d of water were added, and after stirring for an additional 30 minutes, 350 d of DEAE-5ephadex A
-25 (HCOm- type) and eluted with 4 tons of linear grab 4 end (0→α2M) of triethylamine carbonate buffer y- (pH 7,5).The main beak elution number was evaporated to dryness under reduced pressure. Then, it was azeotroped with methanol to desalt it.The yield was 55%.

Phosphorylation can also be performed as follows.

5- L 2 mmol of phosphorous acid 7-sulfochloride, Z 4 mmol of (1-H) in anhydrous dioxane
-1,2,4-triacylide and 2.0 mmol
of triethylamine and heated at 10 to 15°C for 30 minutes.
The mixture was stirred at room temperature for 1 hour. To this was added 1 mmol of zl, sl-guideoxyclidine obtained in section (1-5), and the mixture was stirred at room temperature for 1 hour. Under water cooling, water 51
rll was added and the reaction was stirred at room temperature for 18 hours. The reaction solution was purified by silica gel column chromatography eluting with a step gradient of chloroform-methanol. Dowex 50 (Na” type)
The mixture was stirred in 10 rnl of water at room temperature for 4 hours to convert it into a sodium salt. The yield was 55%.

Triphosphorylation of 2',3'-guideoxyclidine-5'-mono7o7ate was performed by the method of D. E. Hoard et al. (J. Am. Chem. Soc., 87(8).
1,1785 (1965)).

Anhydrous triglylammonium salt α1 of zL, sL-guideoxyclidine-51-mono7-mole7ate obtained in section (1-6)
MF) Solution 11RI! , 1.1'-force 71/dc
Shiimiguzol (L 5 mmol solution in DMF 11
the law of nature! was added and reacted at room temperature for 5 hours. After adding agmmol of methanol and stirring at room temperature for 30 minutes,
Triglylammonicum pyro 7mose 7ate α5mmo
1 of DMF solution 5- was added and stirred at room temperature overnight. The generated precipitate was washed with DMF, treated with an equal amount of methanol together with the supernatant, and concentrated under reduced pressure. Wl residue 601
nl DEAE-5ephadexA-25 (HC
O,- type) and linear gradient end of triethylamine carbonate buffer (pH 7.5) (0-(L)
The eluted portion of the ◎ main peak eluted at 1 t of 4M) was dried under reduced pressure, and then azeotroped with methanol to desalt it. The yield was 75%.

Synthesis of clidine-5'-tri7-osthate: 5-mercurylated-zl,sl-guideoxyclidine-5'
-) The method of Dale et al. (Biochemistry, 14,
2447-2457 (1975)).

1 mmol of zl, a/-gideoxyclidine-5'-tri7-mole7ate obtained in section (1-7) was mixed with 100 ml of αIM sodium acetate 7γ-(pH 6,0)! 5 mmol of mercury acetate was added thereto, and the mixture was reacted at 50° C. for 4 hours. Then, add 9 mm to the solution under water cooling.
01 lithium chloride was added, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in ethyl acetate oil field liquid was measured using 4,4'-bis(dimethylaminocothiobenzophenone), the mercury conversion rate of nucleotides was 90 to 95%. Water-cooled ethanol was added to form a precipitate, which was centrifuged to recover the precipitate.The precipitate was washed with water-cooled ethanol, then with diethyl ether I, and then air-dried.

The yield was 93%.

5-mercurylated-g', 3'-guideoxyclidine-5'
-) Allyl amination of 7-mole 7-ate is performed by Bergs
The method of trom et al. (J, Am, Chem, Soc,
, 98.

1587 (1976)).

5-Mercurylated-2L,3L-guideoxyclidine-5'-triphosphate o obtained in section (1-8), 5m
mol of α1M sodium acetate 7y-(pH 10
) 25rId! I understood. To this was added a 2M aqueous solution of allylamine acetate 3-, and then 4-chloride/4Kzicum chalicum (K, PdC1,) α5 m dissolved in water
Added mo 1. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reacting at room temperature for 24 hours, the reaction solution was filtered through α45μ membrane filter No. 1ζ to remove metallic mercury. The filtrate was purified with 100-DEAE-56p.
After charging Hadex A-25 and cleaning it with α1M sodium acetate buffer (pH&Q) 100-, add triethylamine carbonate buffer (1) H7,5).
+7) It was eluted with 1 t of linear gucdient (QIM→α6M). Dry the main beak eluate under reduced pressure,
Then, it was azeotroped with methanol to desalt it.

Next, this eluate was collected and purified using ODS (C-18) reverse phase high performance liquid chromatography using α5M) Liethyl Leamine Carbonate Buff (pi 47.5). The yield was 65%.

zl, 3j-guideoxy-5 obtained in synthesis section 2 (1-9) of
-(3-1mino)-allylcridine-5'-tri7os7ate Q1 mmol was added to αIM1M carbonate paraffer H9.
,0) dissolved in 20d and 20In9/n! NOXR
ITC (7) DMF solution was added to 0.2 mf and stirred at room temperature for 4 hours to react. The reaction solution was heated to 30 d of D.
EAE-5ephadex A-25 (HCOs
- type), and linear glucidient (αIM→I) of triethylamine carbonate buffer (pH 7,5)
M) 5001! ll! It was eluted with The main peak soluble fraction (eluted at a salt concentration of α6 to α7M) was dried to solidity under reduced pressure, and then azeotroped with methanol to desalt. Yield is 55
%Met.

zl,sL-diodeoxy- obtained in section (1-9)
21 d of a DMF solution of 2 mmol of N-hydroxyl succinimide biotin ester α was added to a solution of 1 mmol of 5-(3-amino)-allylcridine-5′-tri7os7ate α in IM sodium carbonate buffer (pH 85). , reacted for 4 hours at room temperature, and then transferred the reaction solution to a 30 d DEAE-Sephadex A
-25 (HCO, - type) and eluted with a linear gradient (α1M-0.9M) 500 me of triethylamine carbonate puff 7y- (pH 7.5). The main beak eluate with a salt concentration of around α6M was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 60%.

Fluorescently labeled or biotinylated zL according to Figure 2.
, 3J-guideoxy-5-(3-amino)-allylcytidine-5'-)! A method for synthesizing 77t sulfate will be explained.

10 mmol of 21-deoxycytidine was dissolved in 100% anhydrous pyridine, 15 mmol of dimethoxytrityl chloride was added, and the mixture was stirred at room temperature for 18 hours. The solution was diluted with 500 ml of chloroform and then extracted with IN sodium bicarbonate. The chloroform phase was dried and evaporated to dryness under reduced pressure. 21-Deoxy-5'-〇-dimethoxytritylcytidine was obtained by silica gel column chromatography and a step gradient of 7-ol-methanol. The yield was 87%.

The synthesis of the guideeoxy compound shown in sections (2-2) to (2-5) IJ below is carried out by the method of Horvitz et al. (J, P
, Horwtz, et al., Nucleo
sides, nucleotides, earthworks, 304
5 (1962)).

Synthesis of: This example (2-1) 2'-deoxy- obtained by IJI
9.5 mmol of methanesulfonyl chloride was slowly added dropwise to a solution of 7 mmol of 5'-〇-dimethoxytritylcytidine in pyridine under cooling with water. After reacting for 24 hours under water cooling, water 1- was added to stop the reaction. Slowly drop this into 8001 nl of ice water,
The obtained precipitate was filtered, washed with water, and air-dried. The pale yellow product was dissolved in a small amount of amethane and slowly added dropwise into 11 ice water, and the colorless precipitate was filtered and collected. The yield was 80%.

Synthesis: 2'-deoxy-3'-O-mesyl-5'-〇-dimethoxytritylcytidine L2 obtained in section (2-2)
To mmo 1, 101 nl of a solution of α24M Calic A-tert-butoxite in dimethyl sulf7oxide was added, and after reacting at room temperature for 1 hour, it was poured with ice water at 250 rIf.
, dripped inside. After neutralization with dilute acetic acid, the precipitate was centrifuged, washed with water, and air-dried. 80 d of benzene was added to 15 d of this dissolved in ethanol, and the mixture was concentrated under reduced pressure to completely remove water by azeotropic distillation. Benzene was further added and the mixture was concentrated under reduced pressure again, and the resulting precipitate was recrystallized from ethanol. The yield was 75%.

(2-4) 2', 3'-guideoxy-22-cytidine C2-3) 5'-〇-dimethoxytrityl-z/, al-guideoxy-2'-cytidinene 1 obtained by JJ
To a 20-mmol chloroform solution was slowly added 11 equivalents of dry hydrogen chloride gas with stirring under water cooling, and about 3
Detritylation was carried out for 0 minutes under water cooling. The generated precipitate was filtered, washed with chloroform, and then recrystallized from benzene-ethanol (90:10 V/V). The yield was 63%.

(2-5) Synthesis of 2', 3'-guideoxycytidine: zL obtained in (2-4'), 3f-guideoxy-
Dioxane solution of 5 mmol of 2'-cytidinene α25-
To this, 115 μm of 10% hydrogen carbon catalyst 1 was added, and the mixture was reduced at 25° C. for 1 hour under 19 L of hydrogen pressure. After the reaction, the catalyst was filtered off, and the filtrate was dried under reduced pressure. The residue was recrystallized from benzene-petroleum ether. The yield was 80%.

The triphosphorylation shown in sections (2-6) and (2-7) below was performed using the method of Ru5sell et al. (J, Org, Ch
According to em, , 8 (1!1.4889 (1969)),
It was done by chemical method.

2',3'-dideoxysilyl 4 obtained in section (2-5)
- 1 mmol of anhydrous tetrahydro7ran (THF)
) 4 ryunol of triimidazole 7 osulfuric oxide and 100 mJ/l of solution 20-
, 1'-carbonylciimiguzole was added, and at room temperature,
The reaction mixture was stirred overnight. Next, triethylamine 2 me and 5 me were added and after further stirring for 300 minutes, 350-DEAE-5ephadex A
-25 (HCOs-type) I and eluted with a linear gradient (0→α2M) 41 of triethylamine carbonate buff 7A (pH 7.5). The main peak elution number was evaporated to dryness under reduced pressure and then azeotroped with methanol to desalt.

The yield was 6196.

Phosphorylation can also be carried out as follows: 12 mmol I of phosphite 7-ochloride, 24 valent 0! in 0 5 me anhydrous dioxane. (1-H)-
1,2,4-) rear sillide and! Ommol of triethylamine was mixed and stirred at 10-15°C for 30 minutes and at room temperature for 1 hour. To this was added 1 mmol of 2',3'-guideoxycytidine obtained in (2-5)]1Jm, and the mixture was stirred at room temperature for 1 hour. This was cooled with water for 5 liters.
Id! was added and the reaction was stirred at room temperature for 18 hours. The reaction solution was purified by silica gel column chromatography eluting with chloro-7-ol-methanol and Steggcdient. Dowex 50 (Na+ type)
and 10- in water at room temperature for 4 hours to convert it into a sodium salt. The yield was 55%.

Triphosphorylation of 21.31-guideoxycytidine-51-mono7o7ate was performed by the method of D. E. Hoard et al. (supra).

(2-6) 21. 1 mmol of anhydrous trigtylammonicum salt α of al-guideoxycytidine-5′-mono7-mole7ate was added to a DMF solution of 1.1′
- DMF solution of 15 mmol of carbodiimidazole
- was added and reacted at room temperature for 5 hours. After adding α8 mmol of methanol and stirring at room temperature for 309 minutes, a DMF solution 5- of triglylammonium pyro7-mole7ate α5 mmol was added, and the mixture was stirred at room temperature overnight. The generated precipitate was washed with DMF, treated with an equal amount of methanol together with the supernatant, and concentrated under reduced pressure. Residue at 60-DE
Charge AE-5ephadex A-25 (HCOs-type) and add triethylamine carbonate paraffer (p
H7,5) linear gradient end (0 → CL4M) 1
It was eluted at t. The main beak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. Yield is 85
%Met.

Synthesis of cytidine-5'-trifsulphate: 5-mercurylated-2',3'-dideoxycytidine-5'
- Triphosphate was prepared by the method of Date et al. (Bioq
Chemistry, 14, 2447-2457
(1975) )) C quasi-series combination JffiL.

1 mmol of 2f, sl-guideoxycytidine-5'-trif sulfate obtained in section (2-7) was dissolved in 100 d1ζ of αIM sodium acetate backer (pHao), 5 mmol of mercury acetate was added thereto, and the mixture was heated at 50°C. The reaction was allowed to proceed for 4 hours. Then, add 9 mm to the solution under water cooling.
of lithium chloride was added, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in the ethyl acetate extract was measured using 4,4'-bis(dimethylamino)thiobenzophenone, the mercury conversion rate of nucleotides was 90 to 95%. Next, 3 times the amount of water-cooled ethanol was added to the aqueous phase to form a precipitate, which was centrifuged to collect the precipitate. This precipitate was washed with water-cooled ethanol and then with diethyl ethyl, and then air-dried. The yield was 90%.

5-Mercurylated-2'13'-dideoxycytidine-5'
- Allyl amination of tri7o7ate is performed by Bergs
The method of trom et al. (J, Am, Chem, Soc, .
98.

1587 (1976)).

5-Mercurylated-zL, 31-guideoxycytidine-5'-tri7mose7ate α5 m obtained in section (2-8)
mol of 91M sodium acetate 7y-(pH 5
,0) was dissolved in 25 ml. Add 3 me of a 2 M aqueous solution of allylamine acetate, then add 5 m of Balajicum chloride (K!PdC1,) α dissolved in 4 d of water.
Added mol. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reacting at room temperature for 24 hours, the anti-E3m solution was filtered through a 0.45μ membrane filter to remove metal mercury. M solution to 100 rne DEAE-5
Charge ephadex Am25 and set it to 91
Sodium acetate buffer of M pH 5,0) 100-
After washing with triethylamine carbonate buffer y-(pH
7,5) linear gradient end (91M-0,6M)
It was eluted with It. The main peak eluted was dried to dryness under reduced pressure, and then azeotroped with methanol to desalt.

Next, this eluate was fractionated and purified by ODS (C-18) reverse phase high performance liquid chromatography using α5M) ethylamine carbonate buffer (pH 7,5).

The yield was 60%.

Synthesis of 2',3'-guideoxy-5 obtained in section (2-9)
-(3-1mino)-allylcytidine-5'-tri7os7ate (l1mmo+) in α1M carbonate buffer (PH
9,0)20-, 20 drops of a DMF solution of EITC 2- was added thereto, and the mixture was stirred at room temperature for 4 hours to react. The reaction solution was purified by 3Qrne DEAE-3eph.
Adex A-25 (HCOs-type) was charged, and a linear gradient (αIM→IM) of triethylamine carbonate buffer (pH 7,5) was applied at 500 mL.
It was eluted in ml. Main beak elution (α6 to α7
eluted at a salt concentration of M) was dried under reduced pressure, and then
The mixture was azeotropically distilled and desalted. The yield was 53%.

2L,3L-guideoxy-5 obtained in section (2-9)
-(3-amino)-allylcytidine-5'-tri
7 male 7 et Qlo blood o
1 of 0. 19f carbonate natrif b, /(tsunoy-(pH8,5) solution 10rnI!l
This is 2 Tnl of a DMF solution of 0.2 mmol of N-hydrothousylsuccinimide biotin ester! Add
The reaction was allowed to proceed for 4 hours at room temperature, and then the reaction solution was treated with 30 d of DEAR-5 ephadex A-25 (HCOs
-ffM) and linear gradient end (αLM) of triethylamine carbonate buffer (pH 7,5).
-(L9M) eluted at 500 me. The main beak melt with a salt concentration of around 0.6M was dried under reduced pressure,
Then, it was azeotroped with methanol to desalt it. Yield is 6296
Met.

According to Figure 3, fluorescence = a or piotin-labeled 2'
, 3'-dideoxy-7-(3-amino)-allyl-
A method for synthesizing 7-diazaguanosine-5'-triphosphate will be explained.

21.3 shown in section (3-1) and (3-2) TA below
The synthesis of 1-dideoxy-7-diazaguanosine was performed using W
Inkeler et al.'s method (J, Org, Chem, 2
6.3119 (1983)) by 7A transfer glycosylation.

(3-1) Synthesis of 2-amino-7-(2',3'-guigue: 3 mmo 1 (D 2-amino-4-methoxy-7
H-pyrrolo(2,3-d)pyrimidine was dissolved in 20 me of benzene-dimethoxyethane (4:1) containing 3 mmol of methyltrioctylammonicum chloride and 20 me of 50% aqueous sodium hydroxide solution! Inside, 30
Stir for a minute to emulsify.

To this, 1-chloro-2,3-guideoxy-5-O-)
Luo-D-erythrobentofuranose t3mmo
A benzene solution of 30 mt' was added dropwise under stirring to cause a reaction. This suspension was mixed with 100 rne of water and 1001 n! of dichloromecune! The organic phase was concentrated under reduced pressure. This was dissolved in methanol 2°m and treated with concentrated aqueous ammonia at room temperature for 24 hours at 0 vacuum vA&JL, and the aqueous phase was adjusted to pH 4 with hydrochloric acid. The precipitated p-toluoic acid was removed by filtration. The filtrate was concentrated to dryness under reduced pressure.

This was suspended in 100 methanol and refluxed for 10 minutes. Inorganic salts were filtered off, and the filtrate was concentrated under reduced pressure. 3 residues
Dissolved in 0-methanol and applied to a silica gel column 100-
and dichlormechun-methanol (95:5
) was eluted. The yield was 45%.

Composition: 2-amino-7-(2
',3'-Guideoxy-β-formerly-erythrohent7
lanocyl)-4-no) *C 7H-pi01ff(
2,3-d) 5 mmol of pyrimidine L to 1 mol of anhydrous toluene
To this were added 2.6 mmol of p-natrichumothiocresolate and 26 mmol of hexamityl 7-year-old rifukutriamide. This was refluxed for 3 hours under rat poison.

50 ml of water was added to the mixture, and the mixture was washed twice with chlorofluoride at 5° each time. The aqueous phase was adjusted to pH 2 with 2N hydrochloric acid,
Furthermore, it was extracted twice with each 30me chloro7olm. After neutralizing the aqueous phase, it was evaporated and concentrated under reduced pressure. This was recrystallized from 10-m water. The yield was 88%.

The triphosphorylation shown in sections (3-3) and (3-4) below was carried out by a chemical method according to Ru5sell et al.'s method (supra) I.

Synthesis of 2J,31-guideoxy-7 obtained in section (3-2)
-To a solution of 1 mmol of deazaguanosine in anhydrous tetrahydro7ane (THF), 4 mmol of triimidazole 7-osfinic oxide and 100 η of 1,
1'-carbonyldiimidazole was added, and the mixture was stirred overnight at room temperature to react. Then triethylamine 2
After adding 5WIl of water and stirring for an additional 30 minutes, 350- DEAE-3ephadex A-2
5 (HCOs-type) and eluted with 4 tons of linear gradient (0→α2M) of triethylamine carbonate buffer (pH 7,5). The main beak elution number was evaporated to dryness under reduced pressure, then azeotroped with methanol,
Desalted. The yield was 48%.

Phosphorylation can also be performed as follows.

12 mmol of phosphorous 7-osphochloride, 14 mmol of (1-H)-1,2 in anhydrous dioxane in 5d
, 4-) riasilide and 20 mmo+ triethylamine were mixed and stirred at 10-15° C. No. 30 and at room temperature for 1 hour. In addition to this, the 21.
31-Guideoxy-7-deazaguanosine 1 mmo
1 was added and stirred at room temperature for 1 hour. This is cooled on ice,
Water 5- was added and the reaction was stirred at room temperature for 18 hours. The reaction solution was purified by silica gel column chromatography (1ζ) and eluted with a step gradient of 7 volumes of methanol. :o Dowex 5Q (Na
+ form) and 10me (D) were converted into sodium salt by stirring in water at room temperature for 4 hours. The yield was 58%.

FL = 21.37-guidioxy-tudeazaguanosine-5
'-mono-7-mole-7-ate trisulfate oxide, D, E,
The method of Hoard et al. (Ichikawa) was used.

DMF of 0.1 mmol of anhydrous triptylammonicum salt of zL, 31-ri-idaixy7-f azaguanosine-5'-mono-7-osthate obtained in section (3-3)
To 11 nl of solution, add 0.
Add 1 d of 5 mmo I of DMF
I let it react over time. 0.8 m of methanol was added, and the mixture was stirred at room temperature overnight. After washing the generated precipitate with DMF,
The supernatant was treated with an equal amount of methanol and concentrated under reduced pressure. The residue was purified with 60-DEAE-5ephadex.
A-25 (HCOs-type) was charged and eluted with 1 t of linear gradient (0→α4M) of triethylamine carbonate puff 7-ar (pH 7.5). Main beak elution 9 was dried to dryness under reduced pressure and then azeotroped with methanol to desalt. The yield was 81%.

zL, sl-guideoxy-7-mercurylated-7-deazaguanosine-5'-tri7os7ate was prepared by the method of Dale et al. (Biochemistry, 14, 2447).
-2457 (1975)).

21.3/-Guidaixie 7 obtained in (3-4)
-Deasaku1nosine-5'-)! 1 mmol of I7t sulfate was added to a 0.1 M sodium acetate bag (p
H6,0) Dissolve 100 g/+c and add 5 mmo
1 of mercury acetate was added, and the mixture was reacted at 50°C for 4 hours.

Thereafter, 9 mmol of lyticum chloride was added to the solution under ice cooling, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in the ethyl acetate extract was measured using 4,4'-bis(dimethylamino)thiobenzophenone, the mercury conversion rate of scleotide was 90 to 9,596.

Next, 3 times the volume of ice-cold ethanol was added to the aqueous phase to form a precipitate, which was centrifuged to collect the precipitate. This precipitate was washed with water-cooled ethanol, then with diethyl ether, and then air-dried. The yield was 96%.

Synthesis: Allyl amination of zL, sJ-guideoxy-7-mercurylated-7-deazaguanosine-5'-tri7o7ate was performed by the method of Bergstrom et al. (J, Am.

Chem, Soc, 98.1587 (1976)).

(3-5) 2L, 3L-Guideoxy-7 obtained from the river
-Mercurylated-7-deadedi-1-nosine-51-trift sulfate α5 mmol was dissolved in 25 dl of 1M sodium acetate puffer (pI'15.0). To this was added 3d of a 2M allylamine acetate aqueous solution, and then chloride/soaked chloride A calicum (K,
5 mmol of PdC1,)α was added.

The IS liquid gradually turned black, and metallic mercury was deposited on the vessel wall. After reacting at room temperature for 24 hours, the reaction solution was filtered using an α45Il membrane filter-1 trowel to remove metallic mercury. The filtrate was added to 100 fnl of DEAE-8 ephadex.
After washing with 100 d of 0.1 M sodium acetate buffer (pH 5,0), it was eluted with 1 t of a linear gradient (αIM→116 M) of triethylamine carbonate buffer (pH 7,5). The main beak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. This eluate was then converted to 0D
S (C-18) reverse phase high performance liquid chromatography,
α5M triethylamine carbonate backer (pH 7,5)
It was fractionated and purified using The yield was 6096.

21,3/-Guideoxy- obtained in section (3-6)
7-(3-Amino)-allyl-7-7′″azoguanosine-51-tri7 phosphate O, 1 mmol was added to αIM
Dissolved in i acid t 7 y - (pH 9,0) 20 me, and 201n9/n! (7) TMRITC
DMF core liquid 2rrLl! was added and stirred at room temperature for 4 hours to react.

The reaction solution was washed with 30 me DEAE-Sephadex.
A-25 (HCO, - type) was charged and eluted with triethylamine carbonate, /77-(pH 7,5) linear gucdiene) ((LIM→IM) 500me).

Main beak elution number (eluted at a salt concentration of α6 to α7M)
was dried under reduced pressure, and then azeotroped with methanol to desalt. The yield was 52%.

al,s/-guideoxy-7 obtained in section xy-7-(3-amino)-allyl (3-6)
-(3-amino)-allyl-7-diazaguanosine-5
'-Triphosphate Q 1 mmol of Q, LM sodium carbonate buffer CpH & 5) solution 101rd! , N-hydroxyl succinimide biotin ester α
Add 2 mmol of H2me dissolved in DMF and incubate at room temperature for 4 hours.
A time-varying reaction was allowed to occur, and then the reaction solution was mixed with 30-DEAE-
3 Ephadex A-25 (HCO, - type) was charged and triethylamine carbonate buffer y- (pH 7
.
It was eluted at 500-.

Dry the main beak melt with a salt concentration of around α6M under reduced pressure.
Then, it was azeotroped with methanol to desalt it. Yield is 71
It was 96.

Moreover, the 7-diazaguanosine derivative may be synthesized using pre-Q1 nucleoside as a raw material.

In the pre-Q1 nucleoside, a primary amine 7 group is bonded to the 7-position carbon via methylene as a spacer, and the 21.3L-position is simply a guideeoxy form, and triphosphate is bonded to the 5'-position. Example 4: Synthesis of fluorescently or biotin-labeled 2' fluorescently or biotin-labeled 2'
, 3'-didaoxy-7-(3-amino)-aryl-7-deazaadenosine-5'-triphosphate will be described.

21.3 shown below from (4-1) to (4-5)
1-Guideoxy-7-deadeadenosine (2',
3'-Guideoxytuperscidin) was synthesized by K, A
The method of nzii et al. (Agr, Biol, Chem,
.

37.345-3481'1973)).

I Q mmol of tupersidine was suspended in 20 (1 ml of anhydrous pyridine, to which 20 mmol+ dimethoxytrityl chloride was first added at room temperature under stirring, 18
After an hour, 10 mmol of dimethoxytrityl chloride was further added, and the mixture was stirred for an additional 3 days to react. The reaction solution was added to a water-cooled sodium carbonate solution, the product was extracted with chloroform, and the solution was then dissolved under reduced pressure.
It was dried and solidified. This was recrystallized from ethyl acetate. The yield was 33%.

The removal of vicinal diols was performed by the method of E. Vedejs et al. (J. Org. Chem., 39(25), 3641 (
(1974)).

To a pyridine-toluene (1:2) Ml suspension of 1 mmol of 5'-〇-dimethoxytrityl tubercidin obtained in Section (4-1) of this Example, 11 mmol of N,N'-thiocarbonylbisimidazole was added. and stirred at room temperature for 2 hours under nitrogen. The solvent was removed under reduced pressure and the residue was washed with ether. The thiocarbonate of 5'-0-dimethoxytrityl persidine was recrystallized from methanol. The yield was 78%.

The thiocarbonate of 5'-0-dimethoxytrityltuperscidine obtained in section (4-2) was refluxed for 5 hours in 0.7 mmo+isopropyl chloride for 5 hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in 90% ethanol. Zinc dust 12 was added thereto, and the mixture was stirred at room temperature for 18 hours. The precipitate was removed and the solution was evaporated to dryness under reduced pressure and recrystallized from methanol. The yield was 33%.

Into the dioxane solution 25 of 5 mmol of 5'-〇-dimethoxytrityl-27, 3/-dideoxy-2'-7-zoazaadenosinene α obtained in section (4-3),
115ff1g of a 10% radium-carbon catalyst was added, and the mixture was reduced at 25°C for 1 hour under 1 atmosphere of hydrogen pressure. After the reaction, the catalyst was filtered off, and the solution was dried under reduced pressure. The residue was recrystallized from benzene-petroleum ether. The yield was 80%.

To a solution of 1 mmol of 5'-〇-dimethoxytrityl-21.31-dideoxy-2'-7-deazaadenosine in chloroform obtained in section (4-4), 11 equivalents of dry chloride was added under water cooling. Hydrogen gas was slowly added under stirring, and detritylation was carried out for about 30 minutes under water cooling. The produced precipitate was filtered, washed with chloroform, and then recrystallized from benzene-ethanol (90:10 v/v). The yield was #'16796.

The triphosphorylation shown in sections (4-6) and (4-7) below was carried out by a chemical method according to the method of Ru5sell et al. (published in 1lfJ).

Synthesis of adenosine-5I-monophosphate: 21,3'-dideoxy-7 obtained in section (4-5)
-Deazaadenosine To 1 mmol of anhydrous tetrahydrofuran (THF) solution 20TR1, 4 mmol of triimidazole phosphinic oxide and 100 to 1,1'-carponyl diimigul were added, and at room temperature, 1
The reaction mixture was stirred overnight. Then triethylamine 2
- and 5 d of water were added, and after stirring for an additional 30 minutes,
350d DEAE-5ephadex A-25(H
CO3- form) and triethylamine carbonate buffer (pH 7,5) OIJ 27 grade 4 x-:/
) (0+(L 2 M )41!).

The main peak eluate was evaporated to dryness under reduced pressure and then azeotroped with methanol to desalt. The yield was 58%.

Phosphorylation can also be carried out as follows.

5I! ! 12 mmol of phosphorous 7-ochloride, Z 4 mmol of (l-
H)-1,2,4-) riazolide 20 mmol
of triethylamine and heated at 10-15°C for 30 minutes.
The mixture was stirred at room temperature for 1 hour. To this was added 1 mmol of 2',3'-guideoxy-7-7' azaadenosine obtained in section (4-5), and the mixture was stirred at room temperature for 1 hour. To this, 5I11/ of water was added under water cooling, and the reaction solution was heated to room temperature at 18
Stir for hours. The reaction solution was subjected to silica gel column chromatography eluting with a step gradient of chloroform-methanol to prepare i. Dowex50
(Na"u) and IGmt' in water at room temperature for 4
(Stir for hours) Converted to IJ Kum salt. yield! -t
It was 53%.

Synthesis of 2',3'-didaix-7-dadeguanosine-5
Triphosphorylation of '-monophosphate is D, E, H
This was carried out by the method of Oard et al. (mentioned above).

2',3'-Guideoxy-7 obtained in section (4-6)
Anhydrous trigtylammonicum salt of deazaadenosine-5'-monophosphate 1,1'-lupodiimiguzole/l/0.
5 mmol of DMF solution 1- was added and reacted at room temperature for 5 hours. Add 0.8 mmol of methanol, 30
After stirring at room temperature for a minute, 5 d of a DMF solution of 5 mmol of triptylammonicum pyrophos 7ate Q was added.
Stir overnight at room temperature. After washing the generated tj: lees with DMF, the supernatant was treated with an equal amount of methanol and concentrated under reduced pressure. 60d DEAE -5epb of residue
It was charged to adexA-25 (HCO, - type) and eluted with triethylamine carbonate buffer (pH 7,5) and a gradient (O→α4M) 3/. The main peak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. The yield was 81%.

2/,3/-guideoxy-7-mercurylated-7-7''adeadanosine-5'-tri7 phosphate was synthesized according to the method of Date et al. (supra).

21.31-Guideoxy-7 obtained in section (4-7)
-Daadeade/Shin-5'-Triphous 7ate 1mm
ol (LIM Sodium Acetate/4Tfuy-(pH 6,
0) 100d, 5 mmol of mercury acetate was added thereto, and the mixture was reacted at 5Q'C for 4 hours. Thereafter, 9 mmol of lyticum chloride was added to the solution under water cooling, and the mixture was extracted several times with ethyl acetate to remove mercuric chloride. When the amount of mercury in the ethyl acetate extract was measured using 4,4'-bis(dimethylamino/)thiobenzophenone, the mercury conversion rate of nucleotides was 90P-95%. Next, three times the amount of water-cooled ethanol was added to the aqueous phase to form a precipitate, which was centrifuged to collect the precipitate. This precipitate was washed with water-cooled ethanol, then with diethyl ether, and then air-dried. The yield was 89%.

Synthesis of nosine-5'-triphosphate: 21. Allyl amination of 31-guideoxy-7-mercurylated-7-deazaadenosine-5'-triphos7ate was carried out according to the method of Bergstrom et al. (supra). Ta.

5 mmol of 2/, a/-guideoxy-7-mercurylated-7-deazaadenosine-5'-triphos 7ate α obtained in section (4-8) was added to a 0.1 M sodium acetate puffer (pH 5,0 )25TR1. To this was added 2M allylamine acetate aqueous solution 3W11, then 4Tn! Chloride dissolved in water / (Radicum caricum)
K, PdC14) 0.5 mmol was added. The solution gradually turned black, and metallic mercury precipitated on the walls of the vessel. After reacting at room temperature for 24 hours, the reaction solution was filtered with an α45μ membrane filter to remove metal mercury. 10 filtrate
0m1' DEAI knee 5ephadex A-25
After washing with 100 d of 1 M sodium acetate puffer (pH 5,0), wash with triethylamine carbonate buffer (pH 7,5) and glue powder (α1M → 0.6 M) IJ. It was eluted. The main beak eluate was dried under reduced pressure and then azeotroped with methanol to desalt it. This eluate was then reduced to 0
0.5M triethylamine carbonate buffer (pH 7,
5) was used for fractionation and purification. The yield was 61%.

21.31-Guideoxy-7 obtained in section (4-9)
-(3-amino)-allyl-7-deazaadenosine-5
'-Triphosphate α1 mmol was dissolved in 20-91M carbonate buffer (pH 9,0), and 20 fn
A DMF solution of FITC f//d 2- was added and stirred at room temperature for 4 hours to react. The reaction solution was heated to 30-D
E A E -5 ephadex A-25 (HCO3-
) and add triethylamine carbonate buffer y.
(pH 7,5) linear gradient (αIM → IM
) 500-. Main beak elution (α6
-0.7 M salt concentration) was dried under reduced pressure and then azeotroped with methanol to desalt. Yield #″i58%
21.3/-guideoxy-7 obtained in section (4-9)
-(3-amino)-allyl-7-7'azoadenosine-
111M of 5'-triphos7ate 0.1 mmol
Sodium carbonate puffer (pH 8,5) flod
To the N-hydroxylsuccinimidopiotin ester, 2 mmol (i.
- DEAE-5ephadex A-25 (HCO,
- type) and linear gradient end of triethylamine carbonate buffer (pH 7,5) (0,1M → 0
．． 9 M) was eluted at 500 d. Salt concentration α6
The main peak elution fraction near M was dried to dryness under reduced pressure, and then azeotropically distilled with methanol-I to desalt. Yield #'i was 63%.

Example 5: Fluorescent labeling of DNA Example 4. 2/, a/-guideoxy-7-(3) fluorescently labeled with FITC obtained in section (4-10)
DNA was labeled using -A3)-allyl-7-zoadeadanosine-5'-trifuose 7ate under the following conditions.

100mM sodium cacodylate (pH 7.2).

8mM MgC1, 1mM 2-mercaptoethanol, 100μP/dDNA and α5mM FITC
Labeled -21,31-gideoxy-7-(3-amino)-allyl-7-diazaadenosine-5'-triphosphate was added with 1 unit of terminal dixin nucleotide 72lase. The reaction was carried out at 37°C for 1 hour to bind the fluorescently labeled guideeoxy group to the 3'-end of the DNA. After the reaction, Sephadex G
Fluorescently labeled DNA was separated by gel filtration at 100 °C.

From the fluorescence intensity of FZTC, it was found that one DNA molecule was fluorescently labeled.

Example 6: Piotin labeling of DNA Example 4. # by piotine obtained in section (4-11)
DNA was labeled using A-labeled 2/, 3/-gideoxy-7-(3-amino)-allyl-7-deadeenosine-5'-triphosphate under the following conditions.

100mM sodium cacodylate (pH 7.2).

8mM MgC11, 1mM 2-mercaptoethanol, 100p f! /d DNA and 0.5mM
Biotin-labeled-2',3'-guideoxy-7-(3
-Amino)-allyl-7-diazaadenosine-5/-triphosphate was added with 1 unit of terminal dioxynucleotidyl trans7-elase and incubated at 37°C for 1 hour.
A time reaction was performed to bind piotin-labeled guideeoxy groups to the 3'-end of the DNA. After the reaction was completed, the piotin-labeled DNA was separated by gel filtration using Sephadex G100.

Piotin-labeled DNA was transferred onto a nitrocellulose filter at 1 py, 10 py, 100 pP, iny, I
ony, 100n7, and 1 μ2 were spotted and fixed, and the detection degree was examined using a pyotin-avidin detection kit manufactured by Amajam. As a result, 10p
For those that spotted $F or more, I could clearly distinguish between Y/J, but for those that spotted 1 pP, it was barely 'I'l.
Therefore, the detection sensitivity was estimated to be several py/spot.

Guidaoxy reaction for sequence analysis using fluorescent labels was performed under the following conditions. The fluorescently labeled dideoxy compound used was XRITC-labeled ddU obtained in Example L (1-10).
TP (hereinafter referred to as ddUTP+) implementation ff1J 2.
(2-10) Tem et hfc EITCe:4 knowledgedd
CTP (hereinafter referred to as ddCTP Kaoru) Example 3. (3-
TMR [TC-labeled ddde'GTP (hereinafter referred to as "ddGTP" 4B) obtained in 7)] FITC-labeled ddc'ATP (hereinafter referred to as ddAT) obtained in Example 4. (4-10)
P'') was used.

The reaction conditions are as follows.

M13mp single-stranded DNA 2pmol dissolved in 5μm 10mM) Lis-HCl (pH 7.5) - α1mM
10 times more concentrated polymerase in EDTA aqueous solution! 1
1 solution (70mM) lithium hydrochloric acid (I)H7,5),
200mM NaCl, 70mM magnesium chloride, 1mM EDTA) was added to 1pP1 primer 2 pmo+ (1μe) with the sequence shown in Table 3, and incubated at 60°C for 20 minutes.
After heating for several minutes, the mixture was allowed to return to room temperature (required approximately 15 minutes). Add 2μg of this solution to 2μe1 of the mixture of all compositions shown in Table 1.
Polymerase Klenow fragment 1μ! ! (2 units) was added, and after reacting at 37°C for 20 minutes, 1 μe of a mixed solution having the composition shown in Table 2 was added, and the reaction was further carried out at 37°C for 20 minutes. A 90% formamide aqueous solution (
6 μe of stop solution) was added and heated at 95° C. for 3 minutes to completely stop the reaction and form a single strand.

Table 1 Reaction solution 1 ddUTP" 250μM dTTP 4μM ddCTP" 25μM dCTP 10μM ddGTP" 80μM dGTP 4μM ddATP" 125μM dATP 4μM Tris-HCl (pH 7,5) 10mME D
T A 0.1 m M Table 2 Reaction solution 2 dTTP 1mMdcTP
1mM dGTP
1mM dATP 1mM Table 3
Primer sequence (15mer) 5' A G T CA CG A CG T T G
TA 3' Example 8 Sequence analysis The reaction solution obtained in Example 7 was charged to an 8M urine, $-8% polyacrylamide electrophoresis gel using the apparatus shown in Figure @5, and electrophoresed to detect fluorescence. Analyzed by photometer.

A portion of the turns of each fluorescent substance present on the polyacrylamide gel separated by electrophoresis is shown in FIG.

Using an argon laser as a light source, the fluorescent material present on this gel was irradiated with FITC at 488 nm for the light source filter and 520 nm for the light receiving section.
That is, EIT A with the filter of the light receiving part at 570 nm.
C, that is, C, and the filter of the light source section is 512 nm.
and TMRIT the light receiving part filter at 601 nm.
C, ie, G, and XRI TC, ie, T, were recognized using the filter of the light receiving section at 615 nm.

With the above combination, it was possible to efficiently distinguish and detect four types of bases. A part of it is shown in Figure 6,
By processing the chart of each signal corresponding to each of G, C, and T by a combinator, A%G, C%T
We were able to read four types of sequences.

In this case, FIG. 6 is a diagram showing an example of DNA fluorescent bands obtained by the present invention.

Figure 7 shows a method for detecting fluorescent signals for base sequencing.
It is a figure which shows an example.

By performing the data processing as described above, it was possible to analyze about 150 bases.

The composition of the solution used in this example (Table 1) is not necessarily optimal, so it goes without saying that by further optimizing the solution composition, it is possible to analyze even more bases.

(Effects of the Invention) The method for determining the base sequence of polynucleotides and/or oligonucleotides by the chain termination method of the present invention is such that under hybridization conditions, the polynucleotides and/or oligonucleotides are complementary to the sequence to be sequenced. a single-stranded nucleotide sequence (primer), four types of nucleotide triphosphates: unlabeled adenine, guanine, cytosine, and thymine (' or cracil), and one or more differently labeled adenine, guanine. , modified nucleotide derivatives of the 48i class of cytosine and thymine (or cracil) are added together in a single reaction vessel, thereby simultaneously performing one or more types of labeling in a single sequencing reaction. Since this is a method for determining the base sequence of polynucleotides and/or oligonucleotides, there is almost no variation in the intensity of the four types of labeling, fewer errors occur in sequence analysis, and the accuracy of sequence analysis is improved. Furthermore, compared to conventional methods, the operation is much more convenient and automation of the device is also possible.

Furthermore, since no radioactive isotopes are used, special equipment for handling radioactive materials is not required, and there is no risk of waste of the used materials.

Furthermore, the method for determining the base sequence of polynucleotides and/or oligonucleotides by the chemical decomposition method of the present invention (c) labels the 3'-ends of the polynucleotides and/or oligonucleotides with a labeled guidaoxynucleotide derivative. This is a method for determining the base sequence of polynucleotides and/or oligonucleotides, which is characterized by subjecting this to a sequencing reaction, which reduces the number of operating steps and shortens the operating time compared to conventional methods. Improves accuracy.

Furthermore, the analysis device for determining the base sequence of polynucleotides and/or oligonucleotides of the present invention includes:
raw materials for colored, fluorescent, chromogenic or ligand-recognizing labeled polynucleotides and/or oligonucleotides, a zone containing an electrophoretic group, means for introducing the labeled polynucleotides and/or oligonucleotides into the zone; and an optical measuring means for monitoring the movement of labeled polynucleotides and/or oligonucleotides in the gel; therefore, the present invention The present invention is suitable for carrying out base sequencing of polynucleotides and/or oligonucleotides by the chain termination method or chemical decomposition method.

Furthermore, an analysis bag W1t for determining the base sequence of the polynucleotide and/or oligonucleotide of the present invention
ri, raw materials for colored, fluorescent, chromogenic or ligand-recognizing labeled polynucleotides and/or oligonucleotides, HP L C1, and the labeled polynucleotides and/or oligonucleotides are
Since this is an analyzer for polynucleotides and/or oligonucleotides, which includes an optical measuring means for monitoring the polynucleotides and/or oligonucleotides as they move through the PLC group, the polynucleotides and/or oligonucleotides can be analyzed by the chain termination method or chemical decomposition method of the present invention.
Alternatively, it is suitable for carrying out oligonucleotide base sequencing methods.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram illustrating the synthesis of labeled-2',3'-guideoxy-5-(3-amine)-allylclidine-5'-triphosphate of the present invention. Figure 2 shows the labeled -21,31-guideoxy- of the present invention.
Figure 3 is a schematic diagram showing the synthesis of 5-(3-amino)-allylcytidine-5'-tri7os7ate.
-(3-amino)-allyl-7-zoadeguanosine-5
1 is a schematic diagram showing the synthesis of '-triphos 7ate. Figure 4 shows the labeling of the present invention -2t,3'-guideoxy-
7-(3-amino)-allyl-7-diazaadenosine-
Figure 2 is a schematic diagram showing the synthesis of 5'-triphosphate. FIG. 5 is a block diagram of an automated analyzer used for determining the base sequence of the polynucleotide and/or oligonucleotide of the present invention. FIG. 6 is a diagram showing an example of DNA fluorescent bands obtained by the present invention. FIG. 7 is a diagram showing an example of detection of fluorescent signals for base sequencing. that's all

Claims (1)

[Claims] 1. A method for determining the base sequence of polynucleotides and/or oligonucleotides by the chain-end method, which comprises: under hybridization conditions, the polynucleotide and/or oligonucleotide, the sequence to be sequenced; a complementary single-stranded nucleotide sequence (primer), four nucleotide triphosphates: unlabeled adenine, guanine, cytosine and thymine (or uracil), and one or more differently labeled adenine; Four types of modified nucleotide derivatives: guanine, cytosine, and thymine (or uracil)
1. A method for determining the base sequence of polynucleotides and/or oligonucleotides, which is characterized by using two reaction vessels and simultaneously performing one or more types of labeling in one sequencing reaction. 2. The method for determining the base sequence of polynucleotides and/or oligonucleotides according to claim 1, wherein the labeling is four different types of labeling. 3. Early modified nucleotide derivatives are labeled-2', 3'
A method for determining the base sequence of a polynucleotide and/or oligonucleotide according to claim 1 or 2, which is -dideoxynucleotide-5'-triphosphate. 4. Claims 1 to 3, wherein the label functions as a colored, fluorescent, luminescent, or ligand-recognizing label.
A method for determining the base sequence of a polynucleotide/and/or oligonucleotide according to any one of the above. 5. A method for determining the base sequence of polynucleotides and/or oligonucleotides by the chain-end method, which uses four types of dideoxynucleotide derivatives in which adenine, guanine, cytosine, and thymine (or uracil) are each labeled with a different labeling substance. In a single sequencing reaction using
A method for determining the base sequence of polynucleotides and/or oligonucleotides, which is carried out independently and selectively. 6. A patent claim in which, after each of the four types of different labels is applied, the mixture of the four types is directly separated by polyacrylamide gel electrophoresis, and each labeled polynucleotide and/or oligonucleotide separated on the gel is detected. A method for determining the base sequence of polynucleotides and/or oligonucleotides according to item 5. 7. After carrying out each of the four different types of labeling, the mixture of these four types is directly separated by HPLC, and HPLC
Claim 5 Detecting each labeled polynucleotide and/or oligonucleotide separated by LC
A method for determining the base sequence of polynucleotides and/or oligonucleotides as described in 2. 8. A method for determining the base sequence of polynucleotides and/or oligonucleotides by chemical decomposition, in which the 3'-end of the polynucleotides and/or oligonucleotides is labeled with a labeled dideoxynucleotide derivative, and then: A method for determining the base sequence of polynucleotides and/or oligonucleotides, which comprises subjecting the polynucleotides to a sequencing reaction. 9. In the method for base sequencing of polynucleotides and/or oligonucleotides by chemical decomposition method according to claim 8, the polynucleotides and/or oligonucleotides are labeled with different labeling substances, and the different labeled Polynucleotides and/or oligonucleotides are used in each of the chemical degradation reactions, parts of said sequencing reactions are combined and electrophoretically separated together in a polyacrylamide gel, and each separated labeled polynucleotide and/or or polynucleotides and/or oligonucleotides detected on a gel.
Or oligonucleotide base sequencing method. 10. In the method for determining the base sequence of polynucleotides and/or oligonucleotides by chemical decomposition method according to claim 8, the polynucleotides and/or oligonucleotides are labeled with different labeling substances,
The differently labeled polynucleotides and/or oligonucleotides are used in each of the chemical degradation reactions, parts of the sequencing reactions are combined and separated together by HPLC, and each separated labeled polynucleotide is and/or a method for determining the base sequence of polynucleotides and/or oligonucleotides by detecting the oligonucleotides by HPLC. 11. An analysis device for determining the base sequence of polynucleotides and/or oligonucleotides,
A zone containing a colored, fluorescent, chromogenic or ligand-recognizing labeled polynucleotide and/or oligonucleotide source, an electrophoretic gel, means for introducing the labeled polynucleotide and/or oligonucleotide into the zone; and an optical measuring means for monitoring the labeled polynucleotide and/or oligonucleotide as it moves through the gel. 12. An analysis device for determining the base sequence of polynucleotides and/or oligonucleotides,
A source of colored, fluorescent, chromogenic or ligand-recognized labeled polynucleotides and/or oligonucleotides, HPLC, and monitoring of the labeled polynucleotides and/or oligonucleotides as they move through the HPLC gel. A polynucleotide and/or oligonucleotide analysis device comprising: 13. The analysis device according to claim 11 or 12, wherein the optical measuring means is a spectrophotometer. 14. The analysis device according to claim 11 or 12, wherein the optical measuring means is a fluorometer. 15. The analysis device according to claim 11 or 12, wherein the optical measurement means is an optical luminescence detector.