JP2649793B2 - Method for producing labeled oligonucleotide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA sequencing method.

[0002]

2. Description of the Related Art DNA (deoxyribonucleic acid) and RN
The development of reliable sequence analysis of A (ribonucleic acid) is one key to the success of recombinant DNA technology and genetic engineering. Used in conjunction with other techniques of modern molecular biology, nucleic acid sequencing allows the genomes of animals, plants and viruses to be broken down and analyzed into individual genes having a defined chemical structure. Since the function of a biological molecule is determined by its structure, determining the structure of a gene is important in finally processing this basic unit of genetic information in a useful manner. If the genes can be isolated and characterized, they can be modified to allow for the production of a gene product (ie, a protein) having properties different from those of the original protein, Can bring about a change. Microorganisms incorporating natural or synthetic genes can be used as chemical "factories" to produce large amounts of trace human proteins such as interferons, growth hormones and insulin. Giving plants genetic information, they can survive in harsh environmental conditions or produce their own nutrients.

[0003] With the development of modern nucleic acid sequencing methods, various techniques have been developed in parallel. One of them is DN
The emergence of a simple and reliable method of cloning small to medium sized strands of A into bacterial plasmids, bacteriophages, or small animal viruses. This made it possible to produce pure DNA in sufficient quantities for chemical analysis. Another development was the completion of a gel electrophoresis method for highly dissociating and separating oligonucleotides according to their dimensions. However, a key concept of development is that a series of size groups of cloned and purified D
The introduction of a method for generating NA fragments. These fragments, as a collection of various lengths, contain the information needed to determine the sequence of the nucleotides that make up the original DNA molecule. Two different methods for generating these fragments are widely used, one of which was developed by Sanger [F Sanger, Es Nicklen and A. R. Coulson, Proceeding National Academy Sciences USA,
74, 5463 (1977) and J.J. Smith, Method in Enzymology, 65: 56-580 (1980)], and another developed by Maxam and Gilbert. What was done [AM Maxham and W. Gilbert, Methods in Enzymology, No. 65]
Vol. 499-559 (1980)].

[0004] The method developed by Sanger is called the dideoxy chain termination method. In the most commonly used variant of this method, the DNA fragment is cloned into a single-stranded DNA phage, for example M13. These phage DNAs contain DNA polymerase I
Can be used as a template for the prime synthesis of complementary strands by the Klenow fragment. The primer is either a synthetic oligonucleotide or a restriction fragment, the original recombinant DNA that hybridizes specifically to a region of the M13 vector near the 3 'end of the cloned insert.
Isolated from. In each of the four sequencing reactions, prime synthesis is performed in the presence of a sufficient amount of the dideoxy derivative of one of the four possible deoxynucleotides, so that chain growth is dependent on these "dead-end" Are terminated randomly by incorporating nucleotides.
The relative concentration of the dideoxy form relative to the deoxy form is adjusted to produce a length corresponding to all possible chain lengths that can be separated by gel electrophoresis. The products from each of the four prime synthesis reactions are separated by electrophoresis on individual tracks (lanes) of a polyacrylamide gel. An autoradiograph of the DNA pattern in each electrophoresis track is generated using the radiolabel incorporated into the grown strand. The sequence of deoxynucleotides in the cloned DNA is determined by analyzing the band patterns in the four lanes.

The method developed by Maxam and Gilbert chemically purifies purified DNA to produce a series of dimensions of DNA fragments similar to those obtained by the Sanger method. Single stranded or double stranded DNA, labeled at either the 3 'or 5' end with radioactive phosphate, can be sequenced by this method. In the four reaction groups, one or two of the four nucleotide bases are cleaved by chemical treatment. Cleavage is 3
Includes a step process. Modification of the base, removal of the modified base from the sugar component, and strand cleavage in the sugar component. Reaction conditions are adjusted so that most of the end-labeled fragments are of a size (typically 1 to 400 nucleotides) that can be separated by gel electrophoresis. Electrophoresis, autoradiography and pattern analysis are performed in much the same way as the Sanger method. (Chemical treatment necessarily produces two DNA fragments, but only those with end labels are detected on the autoradiograph).

[0006] These DNA sequencing methods are widely used and each have several variants. In each case, the length of the sequence obtained from one reaction group is limited mainly by the resolution of the polyacrylamide gel used for electrophoresis. Typically, 200-400 bases can be decoded from a single gel track. Both of these methods work, but have significant drawbacks. This is a problem mainly associated with electrophoresis. One problem is that it is necessary to use a radioactive label as a label to determine the location of the DNA band on the gel.
The short half-life of phosphorus-32, the instability of the radiolabel, must be considered, as well as radioactive waste and handling issues. More importantly, the nature of the autoradiography (the film image of the radioactive gel band is wider than the band itself) and a comparison of the band positions between the four different gel tracks (which are uniform in terms of band migration) Behavior or not) limits the observed band separation, ie, the length of the sequence that can be read from the gel. In addition, track-by-track irregularities make it difficult to automatically read autoradiographs, and at present these irregularities are better corrected for the naked eye than with a computer. Autoradiographs are time-consuming, tedious and error-prone due to the need to artificially "decode" them. Furthermore, it is impossible to decipher the gel pattern while actually performing the electrophoresis, and the electrophoresis cannot be terminated even when the dissociation is insufficient to separate adjacent bands. The electrophoresis must be terminated at a normalized time and wait for the radiograph to develop before starting sequencing.

[0007]

It is believed that the present invention solves these and other problems associated with the electrophoresis step in DNA sequencing and provides a significant advance in the art.

[0008]

Means for Solving the Problems The present invention relates to a method for producing a labeled oligonucleotide fragment, the method comprising:
Primer labeled with a chromophore or a fluorochrome
A step of hybridizing with an oligonucleotide sequence complementary to the primer, and a step of extending the hybridized primer.

Preferably, the labeled primer is bound to a chromophore or a fluorescent substance via an amino bond.

Preferably, at least one of A, C, G
And T sequencing reactions. More preferably, it is a process for the production of labeled oligonucleotides comprising all four sequencing reactions A, C, G and T, wherein said labels are distinguishable from one another by the spectral properties of said labels. Furthermore, the present invention relates to a novel method for electrophoretic analysis of DNA fragments generated during a DNA sequencing operation, wherein the DNA generated by the sequencing operation using a group of four types of coloring agents or fluorescent agents. The fragments are labeled and can be detected and characterized as they are separated by gel electrophoresis. This detection uses an absorbance or fluorometer that can monitor the labeled bands as they move through the gel.

The present invention further includes a novel DNA fragment analyzer (system) for analyzing oligonucleotide fragments, which is labeled with a chromophore or a fluorescent dye. The source of the oligonucleotide (the chromophore or fluorophore is distinguishable by its spectral properties), separation means capable of separating the oligonucleotide fragments by their size to a high degree, and the labeled Means for detecting fragments.

Preferably, the means for separating the oligonucleotide fragments has a zone having electrophoresis means, and means for introducing the labeled oligonucleotide fragments into the zone.

[0013] Preferably, the apparatus for detecting the labeled fragment includes an optical means for monitoring when the fragment is separated. A preferred optical means is an absorptiometer or a fluorimeter.

Further, the labeled fragment is a fragment obtained by an extension reaction of a primer labeled with a chromophore or a fluorochrome, and the labeled fragment is A,
A device that results from the sequencing reaction of at least one, or at least two, or all four of C, G and T, and is distinguishable from other fragments by the spectral properties of the label.

Furthermore, an apparatus is preferred in which the source of the labeled oligonucleotide fragment is located at one end of a zone and the detecting means is arranged close to the other end of the zone.

The present invention also relates to an apparatus for determining an oligonucleotide sequence, the apparatus comprising: a source of an oligonucleotide labeled with a chromophore or a fluorescent chromophore; A device capable of separating the oligonucleotide fragment by one base according to its size, and a means for detecting the labeled fragment.

The present invention also relates to a method for producing a DNA fragment labeled with a chromogenic or fluorescent label (these fragments are labeled differently), a region containing an electrophoresis gel, and a labeled DN.
A means for introducing the A fragment into the region, and a photometric means for monitoring or detecting the labeled DNA fragment when the labeled DNA fragment moves through the gel and is separated thereby.

It is an object of the present invention to provide a novel method for analyzing sequence of DNA.

Another object of the present invention is to provide a novel DNA fragment analyzer (system).

In particular, it is an object of the present invention to provide an improved method for sequence analysis of DNA.

These and other objects and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings.

Labeled primer for use in the Sanger method
Including methods based on chromatography dideoxy chain termination method of designing Sanger, in the conventional methods of DNA sequencing,
All bands are identified on the gel using a single radiolabel, Phosphorus-32. This method requires that the fragments resulting from the four synthesis reactions be run on separate gel tracks, which creates a problem associated with comparing band mobilities on different tracks. This problem is solved in the present invention by using a group of four color formers or fluorescent color formers, each having a different maximum absorption or fluorescence. Each of these labels is used to chemically couple to a primer to initiate the synthesis of fragment strands. The individually labeled primers are then combined with one of the dideoxynucleotides and used in a prime synthesis reaction with the Klenow fragment of DNA polymerase.

The primer must have the following properties: (1) free 3 'to extend the chain with a polymerase;
Must have a hydroxyl group. (2) It must be complementary to the 3 'unique region of the cloned insert.

(3) It must be long enough to hybridize to form a unique stable duplex.

(4) The chromophore or the fluorochrome must not prevent hybridization or 3 'end extension by polymerase.

Conditions 1, 2 and 3 above are satisfied by several synthetic oligonucleotide primers commonly used for Sanger-type sequencing using the M13 vector. One such primer is a 12mer 5 'TCA
CGA CGT TGT 3 'where A, C,
G and T represent four different nucleotide components of the DNA, A for adenosine, C for cytosine, G for guanosine, and T for thymidine.

The attachment of a chromogenic or fluorescent label is described in US patent application Ser. No. 565,010, filed Dec. 20, 1983, the disclosure of which is incorporated herein by reference. The method used is to introduce an aliphatic amino group at the 5 'end as the last addition in the synthesis of the oligonucleotide primer. This reactive amino group can then be readily coupled with a wide variety of amino-reactive colorants and / or fluorescers. This method is suitable for a labeled primer according to the above condition 4.

The four dyes used must have a high extinction coefficient and / or a relatively high fluorescence quantum yield. In addition, they must have sufficiently far maximum absorption and / or maximum emission. Representative of the four amino-reactive dyes of this type are:

[0029]

[Table 1]

The dyes are bound to the M13 primer and the conjugate is subjected to electrophoresis on a 20% polyacrylamide gel. The labeled primer can be detected by its absorption and fluorescence in the gel. 4
The species of labeled primers all have the same electrophoretic mobility. Dye-bound primers retain the ability to specifically hybridize to DNA, as indicated by their ability to displace underivatized oligonucleotides commonly used in sequencing reactions.

D for use with the Maxam / Gilbert method
In DNA sequencing by the NA-end labeled Maxam / Gilbert method, the ends of DNA fragments having the sequence to be determined must be labeled. This is conventionally done enzymatically with radionucleotides. To use the Maxam / Gilbert method in combination with the dye detection method disclosed in the present invention, the DNA fragment must be labeled with a dye. One way in which this can be done is shown in FIG. Certain restriction endonucleases produce 3 'overhangs, known as the products of so-called DNA cleavage. These enzymes produce a "sticky end", a short extension of single-stranded DNA at the end of double-stranded DNA. This region anneals to the complementary portion of the DNA and is covalently bound by the enzyme ligase into double-stranded DNA. In this way, one of the DNA strands is covalently linked to a detectable component. This component can be a dye, an amino group or a protected amino group, which can be deprotected and reacted with the dye after a chemical reaction.

Sequencing Reaction The dideoxy sequencing reaction was carried out according to the standard method of AJ Smith [Methods in Enchimology, No. 65.
Vol. 56-580 (1980)].
If necessary, a sufficient signal intensity can be given to each band to be detected on an enlarged scale. The reactions are performed using different colored primers for each different reaction, eg, FITC,
EITC is used for the "C" reaction, TMRITC for the "G" reaction, and XRITC for the "T" reaction.The sequencing reaction does not require radiolabeled nucleotide triphosphates.

The Maxam / Gilbert sequencing reaction is carried out by a conventional method (S. F. Gil, Aldrich Himika Acta, Vol. 16 (3), pp. 59-61 (1983)), but one or more end labels may be used. A free or protected amino group that can be any of the four colored dyes or that can later react with the dye.

A part of the gel electrophoresis sequencing reaction is combined and packed into the 5% polyacrylamide column 10 shown in FIG. The relative amounts of the four different reactants in the mixture are adjusted experimentally to give approximately the same fluorescent or absorbing signal intensity from each of the dye / DNA conjugates. This makes it possible to compensate for differences in dye extinction coefficient, dye fluorescence yield, detector sensitivity, and the like.
A high voltage is applied to the column 10, and the labeled DNA fragment is electrophoresed in the gel. Labeled DNA fragments differing by one nucleotide in length are separated by electrophoresis in this gel matrix. At or near the bottom of the gel column 10, the DNA bands are separated from each other and pass through the detector 14 (which will be described in detail below).

The detector 14 detects the fluorescent or chromogenic bands of the DNA in the gel to determine its color and thus the nucleotides corresponding to them. This information is DN
Give the A sequence.

Detection There are many different ways in which labeled molecules separated by length can be detected using electrophoresis on polyacrylamide gels. Hereinafter, four representative methods will be described. 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, (iii) elution of molecules from the gel And detection by chemiluminescence, and (iv)
Detection by absorption of light by molecules. In schemes (i) and (ii), the fluorescence detector must meet the following requirements:
(a) The excitation beam must not have a width that is substantially greater than the width of the band. This is generally in the range of 0.1-0.5 mm. The use of such a narrow excitation beam allows for maximum band separation. (b) changing the excitation wavelength to match the maximum absorption of each of the different dyes, or a single narrow high intensity that excites four fluorogenic agents but does not overlap with any of the fluorescent emissions Light band. (c) The optical device is a photodetector 1
Scattering and reflection for 4 The flux of the excitation light must be minimized. An optical filter that blocks scattered and reflected excitation light is changed with a change in the excitation wavelength. (d) The photodetector 14 must have a fairly low noise level and the emission range of the dye (500 to
(600 nm) should have good spectral response and yield efficiency. (e) The optical system for collecting the emitted fluorescent light must have a high aperture. This maximizes the fluorescent signal. In addition, the depth of field of the condenser lens must include the entire width of the column matrix.

FIG. 3 shows two typical fluorescence detection devices (systems).
And FIG. The device of FIG. 3 is suitable for either single wavelength or multiple wavelength excitation. For single wavelength excitation, filter F4 is one of four bandpass filters that concentrate on the emission wavelength peak of each dye. The filter can be switched every few seconds to continuously monitor each of the four fluorescent agents. In the case of excitation of a plurality of wavelengths, the optical element F3 (excitation filter), DM (dichroic mirror), and F4 (shielding filter) are switched together. In this method, both the excitation light and the observed emission light are changed. The device of FIG. 4 is a good arrangement for single wavelength excitation. This device has the advantage that no moving parts are required, and the fluorescence from all four dyes can be monitored simultaneously and continuously. A third method of detection (iii) is to elute the labeled molecules from the bottom of the gel and bind them to an agent that excites chemiluminescence such as 1,2-dioxyethanedione [S.K. Gil, Andrich Himika Acta, Vol. 16 (3), pp. 59-61 (1983); J. J. Melvin, Liq. Chrom, Vol. 6 (9), Vol. 160
3-1616 (1983)] and the mixture is allowed to flow directly to a detector capable of measuring emitted light at four different wavelengths (this detector is similar to that shown in FIG. 4). , No excitation light source is required). The background signal in chemiluminescence is much lower than in fluorescence,
The result is a higher signal to noise ratio and increased sensitivity. Finally, the measurement can be carried out by measuring the absorbance (iv above). In this case, the variable wavelength beam is
Pass through the gel and measure the decrease in beam intensity based on the absorption of light by the labeled molecules. By measuring the light absorption at different wavelengths corresponding to the maximum absorption of the four dyes,
One can determine which dye molecules were present in the light path. A disadvantage of this type of measurement is that absorption measurements have a substantially lower sensitivity than fluorescence measurements.

The above detection device is connected to a computer 16. At each detection time interval, computer 16 receives a signal proportional to the current measured signal intensity for each of the four colored labels. This information indicates which nucleotides at that point in the observation window
Indicates whether it is the end of the NA fragment. The sequence of the colored band at this point gives the DNA sequence.

The method from labeling to detection and sequence determination will be described together with reference to FIG. FIG. 5 shows the sequencing method by the conventional method and the sequencing method by the improved method of the present invention. Here, a case where four kinds of labels are used will be described as a preferred embodiment. As shown in FIG. 5-II, in the method using radiolabeled DNA, only one kind of label is used, so that one lane (track) is used in polyacrylamide gel electrophoresis.
In this case, four tracks (lanes) corresponding to A, G, C, and T, respectively, are required because it is not possible to distinguish between A, G, C, and T.

On the other hand, in the present invention, since four kinds of distinguishable labels are used, where A, G,
Since it is known whether C and T are present, it is sufficient to use one gel (FIG. 5-III).

Hereinafter, a method for determining DNA using four types of chromophores or fluorescent substances will be described, but this is merely an example and does not limit the present invention.

In the chain termination method, the following steps can be used: (1) Labeling the DNA primer with four chromophores or fluorophores (the labels are mutually distinguishable by their spectral properties) (2) each of the four labeled DNA primers
(3) a step of subjecting each of the reaction solutions of A, G, C and T to a primer extension reaction using dideoxytrinucleotides of A, G, C and T; 4) applying a mixture of the A, G, C, and T reactions to a single gel column to separate DNA; and (5) detecting the separated DNA by coloring or fluorescence. .

In this method, the color or the fluorescence can be visually detected. Therefore, as shown in FIG. 5-II, the DNA sequence is determined by identifying the coloring in order from the bottom of the gel column. When a fluorescent substance is used, the DNA sequence is determined by detecting the maximum value of absorption or excitation. In FIG. 5-IV, the sequence is determined as ACGTGC... By, for example, irradiating four types of excitation light and monitoring them, and arranging them in the order of detection.

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. (2) subjecting each of the four labeled DNAs to a reaction selective for A, G, C, and T, respectively; (3) A, G, C, And (4) applying a mixture of the A, G, C, and T reactions to a single gel column to separate DNA; and (5) And b) detecting the separated DNA by color development or fluorescence.

Detection can be performed in exactly the same manner as in the chain termination method.

The following example illustrates the invention.

[0047] Example 6 shows a block diagram of a DNA sequencing apparatus using one dye at a time. Argon ion laser 1
The beam (4880 °) from 00
Through a polyacrylamide gel tube (sample) 102. Fluorescence excited by this beam is collected by a small F-number lens 106 and passed through an appropriate combination of optical filters 108 and 110 to remove scattered excitation light and use a photomultiplier tube (PMT) 112. To detect. The signal is easily detected on the chart recording paper. DNA sequencing reactions are performed using fluorescein-labeled oligonucleotide primers. The peaks on the chart paper correspond to fragments of fluorescein-labeled DNA of different lengths synthesized in the sequencing reaction and separated in a gel tube by electrophoresis. Each peak contains on the order of 10-15 to 10-16 moles of fluorescein, which is approximately equal to the amount of DNA obtained per band in an equivalent sequencing gel using radioisotope detection. This demonstrates that the fluorescent label is not removed or degraded from the oligonucleotide primer in the sequencing reaction. Furthermore, this indicates that the detection sensitivity is sufficient to perform DNA sequence analysis by this means.

Although the present invention has been described in detail with reference to the embodiments, the present invention is not limited thereto.

[Brief description of the drawings]

FIG. 1 is an illustration of one means of labeling a DNA fragment with fluorescent labels, wherein 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, wherein P
Is a lamp source, L1 is an objective lens, L2 is a condenser lens, F
1 is a UV shielding filter, F2 is a heat cutoff filter, F3 is a band pass excitation filter, F4 is a long pass emission filter, DM is a dichroic mirror, C is polyacrylamide gel, PM
T 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.

[Explanation of symbols]

 10: Column 12: Storage 14: Detector 100: Laser 112: Photomultiplier tube

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location G01N 27/447 G01N 33/50 P 33/50 33/58 A 33/58 27/26 301A 315Z ( 72) Inventor Lloyd M. Smith No. 287, No. 287, Saus Oak Nor, Pasadena, CA, USA Japanese Translation of PCT Application No. Sho 58-502205 (JP, A) , VOL. 7-6! (1979) P.1485-1495 BIOCHEMSTRY, VOL. 16-9! (1977) 1996-2003 BIOCHEM. BIOPHYS. RES. COMMUN. , VOL. 102-3! (1981) p. 1071-1077 MOLEKULL YARNAYA B IOLOGIYA, VOL. 11-3! (1977) p. 598−610

Claims (7)

(57) [Claims] 1. A method for producing a labeled oligonucleotide, comprising the steps of: hybridizing an oligonucleotide fragment labeled with a chromophore or a fluorescent agent with a sequence complementary to the oligonucleotide. And elongating the labeled oligonucleotide fragment. 2. The method of claim 1, wherein the chromophore or fluorescent agent is linked to the oligonucleotide fragment via an amine linkage. 3. The method of claim 1, wherein said labeled oligonucleotide fragment is extendable with a polymerase. 4. The method of claim 3, wherein said oligonucleotide fragment is labeled at or near the 5 ′ end. 5. The method of claim 1, wherein the method comprises a sequencing reaction of at least one of A, C, G and T. 6. The method of claim 1, wherein said method comprises all four sequencing reactions, A, C, G and T, and wherein said labels are distinguishable from each other by their spectral characteristics. 7. The method of claim 1, wherein the hybridizing and stretching steps are part of a sequencing reaction of at least one of A, C, G and T.