JP2649794C - - Google Patents

Description

Description: TECHNICAL FIELD [0001] The present invention relates to an apparatus for analyzing oligonucleotides.
. [0002] The reliability of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)
Is one of the successes of recombinant DNA technology and genetic engineering.
Is the key. When used with other techniques of modern molecular biology, nucleic acid sequences can be determined.
The animal, plant and virus genomes have a defined chemical structure
Individual genes can be broken down and analyzed. The function of biological molecules
The determination of the structure of a gene is determined by this basic unit of genetic information, as determined by its structure
Is important in the final treatment in a useful manner. Isolate and characterize genes
If possible, these are genetics that have different properties than those of the original protein.
Can be modified to allow the production of a progeny product (ie, a protein).
The desired change in the structure of. Natural or synthetic genes
Microorganisms that incorporate are used as chemical "factories" such as interferons,
Produces large amounts of trace human proteins such as growth hormone and insulin
be able to. Give genetic information to plants, survive them in harsh environmental conditions,
Alternatively, it can produce its own nutrients. With the development of modern nucleic acid sequencing methods, various technologies have been developed in parallel.
Have been. One is the simple and reliable cloning of small to medium sized strands of DNA into bacterial plasmids, bacteriophages, or small animal viruses.
It was the advent of a real method. This results in pure DNA in sufficient quantities for chemical analysis.
Production. Other developments have made oligonucleotides available according to their dimensions.
This is the completion of gel electrophoresis for high-level dissociation and separation. However,
The key concept of development is a series of size groups of cloned and purified DNA
The introduction of a method for generating fragments. These fragments, as a collection of their various lengths,
Information needed to determine the sequence of nucleotides that make up the original DNA molecule
Information. Two different methods for generating these fragments are widely used.
One of which was developed by Sanger [F Sanger, E
S Nickren and A. R. Coulson, Proceeding National
Le Academy Sciences USA, Vol. 74, p. 5463 (1977) and
JJH Smith, Method in Enzymology, No. 65
Vol. 56-580 (1980)] and the other is Maxam and Gilba.
(Am Maxam and W. Gilbert)
G, Methods in Enzymology, Vol. 65, pp. 499-559 (19
80)]. [0004] The method developed by Sanger was described as a dideoxy chain terminator.
This is called the law. In the most commonly used variant of this method, a DNA fragment
Is cloned into a single-stranded DNA phage, such as M13. These files
The DNA of the complementary strand by the Klenow fragment of DNA polymerase I
It can be used as a template for prime synthesis. This primer is synthesized
Either an oligonucleotide or a restriction fragment, 3 ′ of the cloned insert
From the original recombinant DNA that specifically hybridizes to the region of the M13 vector near the end
Isolated. In each of the four sequencing reactions, prime synthesis resulted in four possible reactions.
A sufficient amount of one dideoxy derivative of the active deoxynucleotide
As a result, chain growth incorporates these "dead-end" nucleotides.
Is terminated at random. The relative concentration of dideoxy to deoxy is
Lengths corresponding to all possible chain lengths that can be separated by gel electrophoresis
Adjusted accordingly. The products obtained from each of the four prime synthesis reactions are
Separate by electrophoresis on individual tracks (lanes) of the acrylamide gel. Success
Using radiolabels incorporated into the longer strands, the
Create an autoradiograph of the DNA pattern. Data on cloned DNA
The oxynucleotide sequence was analyzed by analyzing the band patterns in the four lanes.
decide. [0005] The method developed by Maxam and Gilbert uses purified DNA.
After chemical treatment, DNA fragments of a series of dimensions similar to those obtained by the Sanger method are obtained.
Generate pieces. Either 3 'or 5' end is labeled with radioactive phosphate
Single or double stranded DNA can be sequenced by this method. 4 types
Of the four nucleotide bases in one or two of the four reaction groups
The treatment causes cleavage. Cleavage involves a three-step process. That is, the base
Modification, removal of the modified base from the sugar component, and
Disconnection. The reaction conditions were such that most of the end-labeled fragments were
To a size that can be separated (typically 1 to 400 nucleotides).
Is adjusted. Electrophoresis, autoradiography and pattern analysis
It is performed almost in the same way. (Chemical treatment necessarily produces two DNA fragments
But only fragments with end labels are detected on the autoradiograph). [0006] These DNA sequencing methods are widely used, and several
It has such a variant. In each case, the length of the sequence obtained from one reaction group was
Limited primarily by the resolution of the polyacrylamide gel used for electrophoresis
. Typically, decoding 200-400 bases from a single gel track
Can be. Both of these methods work, but have significant drawbacks. This is mainly
This is a problem associated with electrophoresis. One problem is the location of DNA bands on the gel.
The use of a radioactive label as a label to determine Phosphorus-3
Two short half-lives, the instability of the radiolabel, must be considered and
Consideration must be given to radioactive disposal and handling issues. More importantly, oh
The nature of the radiography (the film image of the radioactive gel band is
Comparison of the band positions between the four different gel tracks (which are broad)
This may or may not behave uniformly in terms of band movement)
Separation of the resulting bands, ie, the length of the sequence that can be read from the gel, is limited. Further
In addition, the irregularities of each track make it difficult to read the autoradiograph automatically.
Now these irregularities are better corrected by the naked eye than by the computer.
is there. Autoradiographs take time because of the need to artificially “decrypt” them.
It is tedious and troublesome. Furthermore, while actually performing electrophoresis
It is not possible to decipher the gel pattern and to separate adjacent bands
Electrophoresis cannot be terminated even when separation is insufficient, and a certain standardized time
To stop the electrophoresis and develop the radiographs before starting sequencing.
Have to wait. [0007] The present invention relates to electrophoresis in DNA sequencing.
Solving these and other problems with the process and making significant progress in the industry
It is believed to be tricky. [0008] The present invention provides an apparatus for analyzing oligonucleotides.
In this device, the individual oligonucleotides are labeled with a chromophore or a fluorescent dye.
Labeled oligonucleotides obtained by extension reaction of labeled primers
And a plurality of oligonucleotides labeled with the chromophore or fluorescent coloring agent
Separation means capable of separating the compounds according to their size, and the separated label
Detection means for detecting the resulting oligonucleotide with its chromophore or fluorescent chromophore;
An apparatus having: In addition, the present invention relates to the D.C.
A novel method for electrophoretic analysis of NA fragments, comprising the use of four color formers or fluorescent
The group is used to label the DNA fragments generated by the sequencing operation and
Allows for the detection and characterization of fragments as they are separated by electrophoresis. This detection is
An absorbance or fluorometer that can monitor the bands as they move through the gel.
Use The present invention further includes a novel DNA fragment analyzer (system),
The device (system) is a device for analyzing an oligonucleotide fragment, and includes a chromophore or a chromophore.
Is the source of the oligonucleotide labeled with a fluorochrome (the chromophore or the fluorophore)
The color former is identifiable by its spectral properties), a separating means capable of separating the oligonucleotide fragments by their size to a high degree, and the labeling agent.
And a means for detecting the fragment. Preferably, the means for separating the oligonucleotide fragments comprises an electric
A zone having an electrophoresis means; and
Means for introducing into the [0010] Preferably, the means for detecting the labeled fragment comprises a step of separating the fragment.
It is a device that includes optical means for monitoring when it is released. Preferred optical means are
An absorptiometer or a fluorometer. Further, the labeled fragment is labeled with a chromophore or a fluorescent coloring agent.
A fragment obtained by an extension reaction of the primer, and the labeled fragment is
At least one of A, C, G and T, or at least two, or four
, All of which result from the sequencing reaction and depend on the spectral characteristics of the label.
Is a device that can be distinguished from other fragments. Further, the source of the labeled oligonucleotide fragment is
Preferably, the device is located at one end of the zone and the detection means is located close to the other end of the zone.
Good. [0013] The present invention also relates to an apparatus for determining an oligonucleotide sequence.
This device is used for oligonucleotide oligonucleotides labeled with chromophores or fluorochromes.
Source (the chromophore or fluorophore is identifiable by its spectral characteristics)
The oligonucleotide fragment can be separated by one base according to its size.
And a means for detecting the labeled fragment. The present invention also relates to a DNA fragment labeled with a chromogenic or fluorescent
These fragments are labeled differently), the region containing the electrophoretic gel,
Means for introducing the DNA fragment into the region, and the labeled DNA fragment moving in the gel.
Light to monitor or detect the labeled DNA fragment as it is separated thereby
And a degree measuring means. An object of the present invention is to provide a novel DNA sequence analysis method.
. Another object of the present invention is to provide a novel DNA fragment analyzer (system).
Is Rukoto. In particular, it is an object of the present invention to provide an improved method for sequence analysis of DNA.
It is to be. These and other objects and advantages of the present invention will be described with reference to the accompanying drawings.
And will be apparent from the detailed description below. [0019] Designing labeled primers for use in the Sanger method DNA, including methods based on Sanger's dideoxy chain termination method
In conventional methods of sequencing, a single radiolabel, i.e., phosphorus-32, is used.
All bands are identified on the gel. This method uses a group of fragments generated by four kinds of synthesis reactions.
Need to be run on separate gel tracks and bands on different tracks
A problem arises with comparing the mobilities. This problem is addressed in the present invention.
Of four types of coloring agents or fluorescent coloring agents having different maximum absorption or fluorescence, respectively.
This is solved by using groups. Each of these labels is converted to a primer
Used to initiate fragment strand synthesis. Then each
The labeled primer is combined with one of the dideoxynucleotides and used.
And used for a prime synthesis reaction with the Klenow fragment of DNA polymerase. The primer must have the following properties: (1) It must have a free 3 'hydroxyl group to extend the chain with a polymerase.
Must. (2) It must be complementary to the 3 'unique region of the cloned insert.
No. (3) long enough to hybridize and form a unique stable duplex
Must. (4) The chromophore or the fluorescent coloring agent prevents hybridization, or
It should not prevent 3 'end extension by the polymerase. The above conditions 1, 2 and 3 are based on the Sanger-type arrangement using the M13 vector.
Filled with several synthetic oligonucleotide primers commonly used for sequencing
Be done. One such primer is a 12mer 5 'TCA CGA CGT
TGT 3 ', where A, C, G and T are four different nucleotides of DNA
Reotide components are shown, A is adenosine, C is cytosine, G is guanosine, and T is thionine.
Shows midines. The attachment of a chromogenic or fluorescent label is described by the present applicant in 198.
US Patent Application No. 565,010, filed December 20, 3 years,
The disclosure is incorporated herein by reference. The method used is oligonucleotide
Introducing an aliphatic amino group at the 5 'end as the last addition in the synthesis of
And This reactive amino group is then replaced by a wide variety of amino-reactive color formers and
(Or) can be readily coupled to a fluorogenic agent. This method uses the above condition 4
Preferred for labeled primers by The four dyes used have high extinction coefficients and / or relatively high
Must have a fluorescent quantum yield. In addition, the maximum absorption and the
Must have a maximum emission. Representative of these four amino-reactive dyes
The ones are as follows: These dyes are bound to the M13 primer and the conjugate is
Perform electrophoresis on a 0% polyacrylamide gel. Labeled primers
Can be detected by its absorption and fluorescence. 4 types of labeled primers
All have the same electrophoretic mobility. The dye-bound primer is DNA
Retains the ability to specifically hybridize to
Indicated by the ability to replace the underivatized oligonucleotide
. [0029] DNA end labeling for use with the Maxam / Gilbert method In DNA sequencing by the Maxam / Gilbert method, there is a sequence to be determined.
The ends of the DNA fragments to be processed must be labeled. This has traditionally been a radioactive nucleotchi.
This is done enzymatically using Combination with the dye detection method disclosed in the present invention
In order to use the Maxam / Gilbert method, DNA fragments must be labeled with a dye.
No. One way in which this can be done is shown in FIG. Certain restriction endonucleus
Rease is a 3 'overhang known as the product of so-called DNA cleavage.
Generate These enzymes are termed “sticky ends”, ie, the ends of double-stranded DNA.
To generate a short extension of single-stranded DNA. This region is complementary to the complementary portion of DNA.
Neal and is covalently bound by the enzyme ligase to form double-stranded DNA. in this way
Thus, one of the DNA strands is covalently linked to a detectable component. This component
Dyes, amino groups or protected amino groups (which are deprotected after a chemical reaction to
Can be reacted). [0030] Sequencing reaction The dideoxy sequencing reaction was performed by the standard method of AJH Smith [Methods.
In Enchimology, Vol. 65, pp. 56-580 (1980)].
However, if necessary, expand the scale and obtain sufficient signal for each band to be detected.
Can give strength. The reactions are different colored plies for each different reaction.
Reaction, for example, FITC, “C” reaction for “A” reaction
For EITC, for "G" reaction TMRITC, for "T" reaction
Uses XRITC. For sequencing reactions, three radiolabeled nucleotides
Does not require acid. The Maxam / Gilbert sequencing reaction is carried out in a conventional manner [S.F.
Ludrich Himika Acta, Vol. 16 (3), pp. 59-61 (1983)]
The end labeling may be done with one or four colored dyes, or
Is a free or protected amino group that can later react with the dye. [0032] Gel electrophoresis A part of the sequencing reaction was combined to form a 5% polyacrylamide column as shown in FIG.
10 is filled from the storage tank 12 above it. Of four different reactants in the mixture
The relative amounts are adjusted experimentally to give approximately the same fluorescent or absorbing signal intensity from each of the dye / DNA conjugates. As a result, the dye extinction coefficient and the dye
Differences in fluorescence yield and detector sensitivity can be compensated. Column 10
Is applied with a high voltage, and the labeled DNA fragment is electrophoresed in the gel. One nucleotide
The labeled DNA fragments differing only in length are subjected to electrophoresis in this gel matrix.
More separated. At or near the bottom of the gel column 10, the DNA
Are separated from each other and pass through the detector 14 (which is described in more detail below).
Detailed explanation). The detector 14 detects a fluorescent or colored band of DNA in the gel and detects it.
And thus their corresponding nucleotides are detected. This information
Gives the DNA sequence. [0034] detection Detect labeled molecules separated by length using polyacrylamide gel electrophoresis
There are many different ways that can be done. Hereinafter, four representative methods will be described. sand
That is, (i) detection of fluorescence excited by light of different wavelengths for different dyes,
ii) detection of fluorescence excited by light having the same wavelength for different dyes, (ii)
i) elution of molecules from the gel and detection by chemiluminescence, and (iv) light emission by the molecules.
Detection by absorption. In methods (i) and (ii), the fluorescence detector meets the following requirements:
I have to. (a) the excitation light beam has a width substantially larger than the width of the band
No. This is generally in the range of 0.1-0.5 mm. Such a narrow excitation beam
Allows maximum separation of the bands. (b) Change the excitation wavelength
To match the maximum absorption of each of the different dyes, or generate four types of fluorescence
A single narrow high intensity light that excites the agent but does not overlap with any of the fluorescent emissions
It can be a band. (c) the optical device is capable of scattering and
The luminous flux of the reflected excitation light must be minimized. Blocks scattered and reflected excitation light
The optical filter is changed as the excitation wavelength changes. (d) The photodetector 14 is quite
Must have low noise level and the emission range of the dye
Good spectral response and yield efficiency over 500-600 nm per
No. (e) The optical system for collecting emitted fluorescent light must have a high aperture
No. This maximizes the fluorescent signal. Furthermore, the depth of field of the focusing lens is
Must include the full width of the column matrix. Two representative fluorescence detectors (systems) are shown in FIGS. FIG.
The device is suitable for either single wavelength or multiple wavelength excitation. Single wavelength
, The filter F4 has four bands concentrated at the emission wavelength peak of each dye.
This is one of the pass filters. This filter switches every few seconds to generate four types of fluorescence
Each of the agents can be monitored continuously. For multi-wavelength excitation, light
The element F3 (excitation filter), DM (dichroic mirror) and F4 (light-blocking filter)
Switch to the cord. In this method, both the excitation light and the observed emission light are used.
Change people. The device of FIG. 4 is a good arrangement for single wavelength excitation. This
Has the advantage of not requiring any moving parts, and has the advantage of eliminating all four dyes.
Fluorescence can be monitored simultaneously and continuously. The third method of detection (iii above)
) Elutes the labeled molecules from the bottom of the gel and elutes them with, for example, 1,2-dioxyethylene.
Coupled to an agent that excites chemiluminescence, such as tandione (S.K.
Andrich Himika Acta, Vol. 16 (3), pp. 59-61 (1983);
J. J. Melvin, Liq. Chrom, Vol. 6 (9), pp. 1603-1616
(1983)] and a detector capable of measuring emission of the mixture at four different wavelengths.
(This detector is the same as that shown in FIG. 4,
No excitation light source is required). Background signal in chemiluminescence is due to fluorescence
Much lower than that, resulting in a higher signal-to-noise ratio and increased sensitivity
I do. Finally, the measurement can be carried out by measuring the absorbance (iv above). this
In some cases, a variable wavelength beam is passed through the gel and is based on the absorption of light by the labeled molecules.
The decrease in beam intensity is measured. Different waves corresponding to the maximum absorption of the four dyes
Determine which dye molecule was in the optical path by measuring long light absorption
be able to. The disadvantage of this type of measurement is that absorption measurements are inherently lower than fluorescence measurements
Is to have sensitivity. The detection device is connected to a computer 16. At the time of each detection
At intervals, the computer 16 determines the current measurement for each of the four colored markers
Receives a signal proportional to the signal intensity. This information tells you which nucleotide
Indicates whether the end of a DNA fragment of a specific length is observed in the observation window at the time point. The sequence of the colored band at this point gives the DNA sequence. The method from labeling to detection and sequence determination is summarized with reference to FIG.
explain. FIG. 5 shows the sequence determination method according to the conventional method and the improved method of the present invention.
1 shows a sequencing method. Here, as a preferred embodiment, the case where four types of labels are used is described.
And explain. As shown in Figure 5-II, radiolabeled DNA was used.
In this method, only one type of label is used.
In the electrophoresis, in one lane (track), A, G, C, and T
Are indistinguishable, so that 4 corresponding to A, G, C, and T, respectively,
One track (lane) is required. On the other hand, in the present invention, four types of distinguishable labels are used.
To know where A, G, C, and T exist.
It is sufficient to use a tool (Fig. 5-III). Hereinafter, DNA is determined using four kinds of chromophores or fluorescent substances.
The method is described, but is only illustrative and does not limit the invention. In the chain termination method, the following steps can be used: (1) Label the DNA primer with four chromophores or fluorophores (the label may be
(2) each of the four labeled DNA primers is labeled with A, G, C,
For primer extension reaction using dideoxytrinucleotide of T and T
(3) a step of taking and mixing a part of each reaction solution of A, G, C, and T; (4) applying a mixture of the A, G, C, and T reactions to a single gel column , DNA
And (5) detecting the separated DNA by color development or fluorescence. In this method, color or fluorescent light can be visually detected. Therefore
As shown in Fig. 5-II, if the coloring is identified in order from the bottom of the gel column, DNA
The sequence is determined. If fluorescent material is used, the maximum absorption or excitation
By detecting the value, the DNA sequence is determined. Figure 5-IV shows, for example, four types of excitation
By illuminating the light and monitoring it and arranging it in the order of detection, the sequence becomes ACG
T GC... Is determined. The following steps may be used in the chemical degradation method: (1) Label the DNA with four chromophores or fluorophores in the manner shown in FIG.
The labels are distinguishable from one another by their spectral properties); (2) separating each of the four labeled DNAs,
(3) Step of taking and mixing a part of each reaction solution of A, G, C, and T; (4) Mixture of A, G, C, and T reactions Is applied to a single gel column and the DNA is
And (5) detecting the separated DNA by color development or fluorescence. Detection can be performed in exactly the same manner as in the chain termination method.
You. The following example illustrates the invention. [0045] An example FIG. 6 shows a block diagram of a DNA sequencing device using one dye at a time.
You. Beam controller (4880 °) from argon ion laser 100
The solution is passed through a polyacrylamide gel tube (sample) 102 by 104. To this beam
The more excited fluorescence is collected by the lens 106 having a small F-number and
Remove scattered excitation light by passing through the appropriate combination of optical filters 108 and 110
Then, detection is performed using a photomultiplier tube (PMT) 112. Signal is chart recording
It is easily detected on paper. DNA sequencing reactions were performed on fluorescein-labeled oligos.
This is performed using nucleotide primers. The peaks on the chart paper are
Different lengths synthesized in a sequencing reaction and separated in a gel tube by electrophoresis
Fluorescein-labeled DNA fragment. Each peak is 10-15
Contains fluorescein in the order of 10-16 moles, which is
About the amount of DNA obtained per band in an equivalent sequencing gel using
equal. This is because the fluorescent label is the oligonucleotide primer in the sequencing reaction.
Proof that they will not be removed or decomposed. Furthermore, this is
Degree is sufficient to perform DNA sequence analysis by this means.
. As described above, the present invention has been described in detail with reference to examples.
It is not limited to only.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory view of one means for labeling a DNA fragment end with a fluorescent label, comprising PstI
And T4 DNA ligase are enzymes commonly used in recombinant DNA technology. FIG. 2 is a block diagram of an automated DNA sequencing device, ie, a gel electrophoresis device. FIG. 3 is a schematic view of an optical arrangement in a detection device, where P is a lamp source, L1 is an objective lens, L2 is a condensing lens, F1 is a UV blocking filter, F2 is a heat blocking filter,
F3 is a band-pass excitation filter, F4 is a long-pass emission filter, DM is a dichroic mirror, C is a polyacrylamide gel, and PMT is a photomultiplier tube. FIG. 4 is a schematic view of another optical arrangement in the detection device, wherein F1 to F4 are bandpass filters that concentrate on the maximum emission of various dyes, P1 to P4 are photoelectron amplifier tubes, and the excitation light is a filter. The wavelength does not transmit any of F1 to F4. FIG. 5 is a comparison diagram of the conventional method for determining the DNA sequence of the sequence shown in FIG. 1 and the method of the present invention. FIG. 6 is a block diagram of a preferred DNA sequencing device according to the present invention. [Description of References] 10: Column 12: Storage 14: Detector 100: Laser 112: Photomultiplier tube

Claims (1)

Claims 1. An apparatus for analyzing oligonucleotides, the apparatus comprising: means for separating a plurality of labeled oligonucleotide sources, wherein each oligonucleotide is a chromophore or It was obtained at a primer stretched reaction using labeled primers in fluorescent agent; having; Contact and was released該分, detecting means for detecting the labeled oligonucleotide in the chromophore or fluorescent agent ,apparatus. 2. The apparatus of claim 1, wherein said means for separating labeled oligonucleotides comprises: a zone containing an electrophoretic medium; and means for introducing said labeled oligonucleotides into said zone. . 3. The apparatus of claim 2, wherein the means for introducing the labeled oligonucleotide into the zone comprises a source of kinetic power. 4. The apparatus according to claim 1, wherein said separation means is a separation means capable of separating a plurality of oligonucleotides labeled by a difference of at least one base. 5. The means for detecting the separated, labeled oligonucleotides comprises optical means for measuring the spectral properties of the chromophore or fluorescent agent used to label the individual oligonucleotides. The apparatus of claim 1. 6. The apparatus according to claim 5, wherein said optical means is an absorptiometer or a fluorimeter. 7. The method according to claim 1, wherein each labeled oligonucleotide is linked via an amine linkage to
The device of claim 1, wherein the device is associated with a chromophore or a fluorescent chromophore. 8. The apparatus of claim 1, wherein said primer extension reaction is a component of a dideoxy chain termination DNA sequencing protocol. 9. In performing one set (A, G, C, T) of chain termination DNA sequencing reactions, at least one of said labeled primers used in said set is used in another set used in said set. 9. The device of claim 8, wherein the device is distinguished from the labeled primer by its spectral characteristics. 10. A set of chain termination DNA sequencing reactions (A
, G, C, T), wherein the individual labeled primers are distinguished by their spectral properties from other labeled primers used in the set. 11. The apparatus of claim 2, wherein the source of the labeled oligonucleotide is at one end of a zone and the detection means is located proximate the other end of the zone.