JPS61173158A - Method of determining arrangement of deoxyribonucleic acid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to DNA sequencing methods.

DNA (deoxyribonucleic acid) and RNA (ribonucleic acid i1!
The development of reliable sequence analysis methods is one key to the success of recombinant DNA technology and genetic engineering. When 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 with defined chemical structures.

Since the function of a biological molecule is determined by its structure,
Determining the structure of genes is the ultimate way to process this basic unit of genetic information in a useful way! It is. If genes can be isolated and characterized, they can produce gene products with properties different from those of the original protein (
i.e., proteins) can be modified to bring about the desired changes in their structure that allow for the production of proteins. Microorganisms into which natural or synthetic genes have been incorporated can be used as chemical "factories" to produce large amounts of valuable human proteins, such as internases, growth hormones, and insulin.

Plants can be given genetic information to enable them to survive in harsh environmental conditions or to produce their own propagation media.

The development of modern nucleic acid sequencing methods involves parallel developments in various technologies. One of these was the advent of simple and reliable methods for cloning small to medium sized strands of DNA into bacterial plasmids, bacteriophages, or small animal viruses. This allowed the production of pure DNA in sufficient quantities for chemical analysis. Another development was the perfection of gel electrophoresis methods for highly resolving separation of oligonucleotides according to their size. However, an interesting conceptual development was the introduction of a method for producing cloned and purified DNA fragments of a range of sizes, which in collections of various lengths contain the nucleotides that make up the DNA molecule. Contains the information needed to determine the sequence of Two different methods for generating these fragments are widely used, one of which was developed by Sanger [F.
Coulson, Grossifying National Academy of Sciences USA, Vol. 74, p. 5463 (19)
77) and N.J.H. Smith, Mesotsuzu.
In Enzymology, Vol. 65, pp. 56-580 (
(1980)), and the other was developed by Maxam and Gilbert [N.M. Maxa and W. Gilbert, Methods in Enzymology, Vol. 65, pp. 499-559 (1980)]. be.

The method developed by Sanger is called the dideoxy chain termination method. In the most commonly used variant of this method, DNA fragments are converted into single-stranded DNA fragments, such as M13.
Clone into A phage. These phage DN
A can be used as a deterrent for primed synthesis of complementary strands by the Klenow fragment of DNA polymerase II. The primer is either a synthetic oligonucleotide or a restriction fragment isolated from the original recombinant DNA that specifically hybridizes to a region of the M13 vector near the 3' end of the cloned insert.

In each of the four sequencing reactions, prime synthesis is carried out in the presence of a sufficient amount of the dideoxy congener of one of the four possible deoxynucleotides, so that the growing strands are derived from these "original" ends. Optionally terminated by the incorporation of nucleotides K.

The relative concentrations of dideoxy versus deoxydeoxy forms are adjusted to produce terminations that accommodate all possible chain lengths that can be separated by gel electrophoresis. The products from each of the four prime synthesis reactions are then electrophoretically separated in individual tracks on a polyacrylamide gel. Radioactivity incorporated into the growth chain is used to generate a radiograph of the DNA pattern at each electrophoretic track.

The sequence of deoxynucleotides in the cloned DNA is determined by examining the banding patterns in the four trajectories.

The method developed by Maxam and Gilbert
Chemical treatment of NA is used to generate DNA fragments in a range of sizes similar to those obtained by the Sanger method. 3
Single-stranded or double-stranded DNA labeled with radioactive phosphate at either the '' or 5' ends can be sequenced by this method. In four reaction groups, one or two of the four nucleotide bases are cleaved by chemical treatment. Cleavage involves a three step process. namely, modification of the base, removal of the modified base from the sugar moiety, and strand scission in this sugar moiety. Reaction conditions are adjusted so that the majority of S-terminated product fragments are in a size range (typically 1-400 nucleotides) that can be separated by gel electrophoresis. Electrophoresis, radiography and pattern analysis are performed in much the same way as are performed for the Sanger method.

(Chemical fragmentation always yields two pieces of DNA each time it is carried out, but only the piece with the end label is detected in the radiograph).

Both of these DNA sequencing methods are widely used and each have several variations. In each case, the length of sequences obtained from one reaction group is primarily limited by the resolving power of the polyacrylamide gel used for electrophoresis. Typically, 200 to 4
00 bases can be decoded. Although both of these methods are successful, they have significant drawbacks, which are primarily problems associated with electrophoretic methods. One problem is that D in the gel
It is necessary to use a radioactive label as a label to locate the NA band. The short half-life of phosphorus-32, the instability of the radioactive am agent, must be addressed, as well as radioactivity disposal and handling issues. More importantly, the nature of the radiographic technique (the film image of the radiogel band is broader than the band itself) and the comparison of band position between the four different gel tracks (this is important in terms of band mobility) behave uniformly,
(sometimes not) limits the observed band separation and thus the length of sequence that can be read from the gel. Additionally, track-to-track irregularities make automated scanning of radiographs difficult; compensation for these irregularities is currently better with the naked eye than with a computer. This need for artificial "interpretation" of radiographic photographs is time consuming, cumbersome, and error-prone. In addition, it is impossible to decipher the gel pattern while actually performing electrophoresis, so that the electrophoresis can be terminated even when resolution is insufficient to separate adjacent bands. Electrophoresis must be completed at a given time and radiographs must be prepared before sequencing can begin.

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

K required, the present invention can reduce D generated during DNA sequencing operation.
A novel method for electrophoretic analysis of NA fragments in which a group of 4's colorants or fluorophores are used to label DNA fragments generated by sequencing chemistry and to separate them by gel electrophoresis. This detection uses an absorption or fluorometer that can monitor the labeled bands as they move through the gel.

The invention further includes a novel apparatus for analyzing DNA fragments, which apparatus can be used as a source of chromogenic or fluorescently labeled DNA fragments, which fragments can be labeled differently. , a zone containing an electrophoretic gel, and means for introducing the $lI&DNA fragment into said region;
It consists of photometric means for monitoring or detecting the a-identifier DNA fragment as it moves through the gel and is separated from K.

An object of the present invention is to provide a novel method for sequence analysis of DNA.

Another object of the present invention is to provide a new analysis system for DNA fragments.

%, 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 invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings.

Design of labeled primers for use with the Sanger method, including methods based on Sanger's dideoxy chain termination method
In traditional methods of NA sequencing, a single radiolabel,
Thus all bands are identified on the gel using phosphorus-32. This necessitates subjecting the fragment groups produced by the four synthetic reactions to separate gel tracks, which creates problems associated with comparing band mobilities in different tracks.

This problem is particularly solved in the present invention by using a group of 48 color formers or fluorogens, each with a different maximum absorption or fluorescence. Each of these irons is chemically linked to the primer used to initiate the synthesis of the fragment strand. Each flIR primer is then combined with one of the dideoxynucleotides and used in a primed synthesis reaction with the Klenow fragment of DNA polymerase.

The primer must have the following properties: C1) Must have a free 3' hydroxyl group for chain extension by the polymerase. (2) Must be complementary to the unique region 3' of the cloned insert. (31) must be long enough to hybridize to form a unique stable duplex. (4) The chromophore or fluorophore must be long enough to prevent hybridization or to
Do not interfere with the terminal cap length.

Conditions 1.2 and 3 above are M1! It is filled with several synthetic oligonucleotide primers commonly used for vector-based Sanger sequencing. One of the primers for this glaze is t2mers'T CA CG A
CG TT G 'I' 3 ', where A
, C%G and T represent the four different nucleotide components of DNA, A for adenosine, C for cytosine, G for guanosine, and T for thymidine.

The chemistry for attaching chromogenic or fluorescent labels is described in U.S. Pat. do. The method used is to add 5 as the last addition in the synthesis of oligonucleotide primers.
By introducing an aliphatic amino group at the terminal end, the reactive amino group can then be easily combined with a wide variety of amine-reactive color formers and/or fluorogens. This method is suitable for labeled primers according to Condition 4 above.

The 41'li dye used must have a high extinction coefficient and/or a relatively high fluorescence yield.

Furthermore, it must have well-separated absorption and/or emission maxima. Representative 48i amine-reactive dyes of this type are: fluorescein isothiocyanate (FI'l'C).

Ex λmax = 495, λ1 gift = 520. ε4,5sa8×
10), eosin isothiocyanate (EI'rC).

λEx =522,! ”=543, ε-8X1 0'
), tetramethylrhodamine isothiocyanate (TM
RI TC, 2” = 5 5 0, 2” = 57
8, max maxε5
5. :4X10), and #substituted isothiocyanic acid rhodamiy (XRITC, λ""=sao, λ-max
max=604,'sa
. :8X10'), where λ indicates the wavelength (nanometers), Ex is the excitation, Em is the emission, and m
ax is the maximum and e is the molar extinction coefficient.

These dyes are bound to the M13 primer and the conjugates are electrophoresed on a 2ON polyacrylamide gel. The labeled primer is visible by its absorption and fluorescence in the gel. All four labeled primers have the same electrophoretic mobility. The dye-conjugated primers retain the ability to differentially hyperdize DNAk%, as demonstrated by their ability to displace underivatized oligonucleotides commonly used in sequencing reactions.

DNA end-labeling used with the Maxam/Gilbert method In the Maxam/Gilbert method of DNA sequencing,
The end of the DNA piece containing the sequence to be determined must be sampled. This is conventionally done using radioactive nucleosides.
It is done uniformly. In order to use the Maxam/Gilbert method in conjunction with the dye detection scheme disclosed in this invention, the DNA pieces must be labeled with a dye. One way this can be done is shown in FIG. Different types of restriction endonucleases produce what are known as 3' overhangs, such as the products of the so-called DNA Ifi cleavage. These enzymes produce “sticky ends” or double M-stranded DN
Generate a short extension of single-stranded DNA at the end of the A piece. This region is fused with a complementary part of the DNA, which can be covalently linked by the gauze (2]i:DNA). In this way, one of the strands is covalently linked to the detectable component. This component can be a dye, an amino group or a protected amino group (which can be deprotected after a chemical reaction and reacted with the dye).

Sequencing Reaction The dideoxy sequencing reaction was performed using the standard method of N.J.H. Smith [Methods in Etchmology, No. 65].
Vol., pp. 56-580 (1980)); however, the scale can be expanded as necessary to provide a signal intensity equal to that of light in each band for detection. Reactions are carried out using different colored plies for different reactions, e.g. for the rAJ reaction FITC,
EITC for cJ reaction, I[T for J reaction
MRI'l"C, "X)tITc for TJ reaction
is used. Sequencing reactions do not require radiolabeled nucleoside triphosphates.

The Maxam/Gilbert sequencing reaction was performed in a conventional manner (Niss F. Gil, Aldrich Himica Acta, Vol. 16(3), pp. 59-61 (1985)), with the exception that the terminal! s# can be either a single or 4fi colored dye, or a free or retentive amino group that can be subsequently reacted with a dye.

Parts of the gel electrophoresis sequencing reaction were combined to yield 5% as shown in Figure 2.
The polyacrylamide column 10 is filled from the reservoir 12 above it. The relative amounts of the four different reactants in the mixture were determined experimentally to give approximately the same fluorescent or absorbing signal strength from each dye/DNA conjugate.
1g1l is adjusted. This can compensate for differences in K such as dye extinction coefficient, dye fluorescence yield, and detector sensitivity. A high voltage is applied to the column 10 to inject labeled DNA into the gel.
One copy of the fragment μ is electrophoresed. Labeled DNA fragments that differ in length by a single nucleotide are separated electrophoretically in this gel matrix. At or near the bottom of the gel column 10, the DNA bands are dissociated from each other and passed through a detector 14 (described in more detail below).

Detector 14 detects the fluorescent or chromogenic bands of DNA in the gel to determine its color and thus detect their corresponding nucleotides. This conventional method yields DNA sequences.

Detection There are many different ways in which long-separated naked image molecules can be detected using polyacrylamide gel electrophoresis. Four typical methods will be described below. (1) detection of fluorescence excited by light of different wavelengths for different dyes; (11) detection of fluorescence excited by light of the same wavelength for different dyes;
D. Elution of molecules from the gel and detection by chemiluminescence, and detection by absorption of light by the hexamolecule. In methods (1) and (II), the fluorescent detector must meet the following requirements. (a) The excitation magneto-optical must not have a height substantially greater than the height of the band. This is general IC1
It is in the range of 1 to [L5 simple. The use of such a narrow excitation beam allows maximum separation of bands. (b) Varying the excitation wavelength to match the respective absorption maxima of different dyes, or a single narrow high-intensity modulation that excites the four fluorogens but does not overlap any of the fluorescence emissions. Can be used as a light band.

(C) The optical system must minimize the flux of scattered and reflected excitation light to the photodetector 14. Optical filters that block scattered and reflected excitation light are replaced when the excitation wavelength changes. (d) The photodetector 14 must have a fairly low noise level and must have good spectral response and yield efficiency over the entire dye emission range (SOO to 600 nm for the above dyes). (e) The optical system for collecting the emitted fluorescent light must have a high degree of aperture. This maximizes the fluorescent signal. Furthermore, the depth of field of one focusing lens must include the entire width of the column matrix.

Two typical fluorescence detection devices (systems) are shown in FIGS. 3 and 4. The apparatus of FIG. 3 is suitable for either single wavelength excitation or multi-wavelength excitation. For single wavelength excitation, filter F4 is one of four bandpass filters that focus on the emission wavelength peak of each dye. This filter can be switched every few seconds to continuously monitor each of the 4 s of fluorogen. For excitation of multiple wavelengths, the optical element F
3 (excitation filter), DM (dichroic mirror) and F4 (light blocking filter) are switched together. In this method, both the excitation light and the observation emission light are varied. The device of FIG. 4 is a good arrangement for single wavelength excitation. This device has the advantage of not requiring any moving parts and 421
! Fluorescence from all of the dyes can be monitored simultaneously and continuously. A third method of detection (111 above) is to elute the labeled molecules from the bottom of the gel and transfer these to e.g.
．． Combined with an agent that excites chemiluminescence, such as 2-dioxyethanedione [K.
983); G.G. Melvin, Liq-Chro
m%K 6 Volume (9), Pages 1603-1616 (19
85) and flowing this mixture directly into a detector that can measure the emitted light at four separate wavelengths (this detector is similar to the one shown in Figure 4, but requires an excitation light source). ). The background signal in chemiluminescence is much lower than in fluorescence, resulting in a higher signal-to-noise ratio and increased sensitivity. Finally, the measurement can be carried out by measuring the one-absorbency (the above +V
). In this case, a light beam of variable wavelength is passed through the gel and the decrease in beam intensity due to absorption of light by labeled molecules is measured. By measuring the light absorption at different wavelengths of the four willows, which correspond to the maximum absorption of the dye in the four willows, it is possible to determine which dye molecules were present in the optical path. A disadvantage of this type of measurement is that absorption measurements have inherently lower sensitivity than fluorescence measurements.

The above detection device is placed facing the computer 16.

Each time a test is performed, the computer 16 displays seventy-four of four types of colored markers.
Each receives a signal proportional to the current measurement signal strength. This information indicates which nucleotide will now end a DNA fragment of a particular length in the observation window. The alignment of the colored bands at this point gives the DNA sequence.

FIG. 5 shows the type of data obtained by conventional methods and the type of data obtained by the improved method described in the present invention.

The invention is illustrated by the following examples.

EXAMPLE FIG. 6 shows a block diagram of a DNA sequencing device using 1N dye at a time. Argon ion laser
10 beams from 100 (4880 people)
4 into a polyacrylamide gel tube (sample) 102. The excited light from this beam is collected by a fluorescent low f-number lens 106, passed through an appropriate combination of optical filters 10 and 110 to remove scattered excitation light, and a photomultiplier tube (PMT) 112. Detect using.

The signal is easily detected on chart paper.

DNA sequencing reactions are performed using fluorescein-labeled oligonucleotide primers. The peaks on the chart paper correspond to fragments of fluorescein@R 1) NA with different lengths synthesized in the sequencing reaction and electrophoretically separated in gel tubes.

Each peak contains on the order of 10-18 to 10-16 moles of fluorescein, 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 explained in detail using Examples above, the present invention is not limited to these.

[Brief explanation of drawings]

FIG. 1 is an illustration of one means of end-labeling DNA fragments with fluorescent labels; Pst I and T,a DNA ligases are enzymes commonly used in recombinant DNA technology; FIG. is a block diagram of an automated DNA sequencing device, that is, a gel electrophoresis device, and FIG. 3 is a schematic diagram of the optical arrangement in the detection device, where P is a lamp source, Ll is an objective lens, F2 is a condenser lens, and Fl is a UV light blocking filter,
F2 is a heat cutoff filter, F5 is a bandpass excitation filter, F4 is a longpass emission filter, DM is a dichroic mirror, C is a polyacrylamide gel, PMT is a photomultiplier tube, Schematic diagram of the optical arrangement, where F1-F4 are bandpass filters that focus on the maximum emission of different dyes, P1-P4 are photomultiplier tubes, and the excitation light is transmitted through any of the filters F1-F4. (Figure 5 is a comparison of the types of data obtained by DNA sequencing of the sequences shown in Figure 1, and Figure 6 is a block diagram of a preferred DNA sequencing device according to invention #4. This is a diagram. 10: Column 12: Storage tank 14: Detector 100: Laser 112: Photoelectron intensifier tube FIG, 2 FIG, 5) : Nizushi Otsuchi Nanayamara Midino Vl/-1 Piece-
I
71 beg''4 ke) 1''-1/:r Otsuku] \hito Lf-'+ord

Claims (17)

[Claims] (1) A DNA sequencing method using a chain end method, which is characterized in that the primer oligonucleotide is labeled with a colored label. (2) A DNA sequencing method using a chain end method, which is characterized in that the primer oligonucleotide is labeled with a fluorescent label. (3) In the DNA sequencing method using chemical decomposition method, D
DNA characterized by labeling NA molecules with colored labels
Sequencing methods. (4) In the DNA sequencing method using chemical decomposition method, D
DNA characterized by labeling NA molecules with a fluorescent label
Sequencing methods. (5) In the DNA sequencing method using the chain end method, 4
The primer oligonucleotides used in each of the species sequencing reactions A, C, G and T have different colored labels attached thereto, and the parts of the sequencing reactions are combined and electrophoresed together in a polyacrylamide gel. , and a DNA sequencing method characterized in that detection is performed after separation on a gel. (6) In the DNA sequencing method using the chain end method, 4
The primer oligonucleotides used in each of the species sequencing reactions A, C, G, and T have different fluorescent labels attached thereto, and portions of the sequencing reactions are combined and electrophoresed together in a polyacrylamide gel. 1. A method for determining DNA sequence, which is characterized in that detection is performed after separation on a gel. (7) In the DNA sequencing method using chemical decomposition method, D
Labeling NA molecules with different colored labels and different colored DNA
is used in each of the chemical change reactions, parts of the sequencing reactions are combined and electrophoresed together on a polyacrylamide gel, and detected after separation on the gel. (8) In the DNA sequencing method using chemical decomposition method, D
Labeling NA molecules with different fluorescent labels and labeling them with different fluorescent DNA
A DNA sequencing method characterized in that A is used in each of the chemical change reactions, parts of the sequencing reactions are combined and electrophoresed together on a polyacrylamide gel, and detected after separation on the gel. . (9) In the DNA sequencing method using chemical decomposition method, D
A DNA sequencing method characterized by labeling an NA molecule with an amino group and bonding it to a dye molecule after a sequencing reaction. (10) coupling the products of each of the different sequencing reactions with a different colored dye, combining portions of the dye-labeled reactions and electrophoresing them in a polyacrylamide gel;
10. The method according to claim 9, wherein the detection is performed after separation on a gel. (11) In the DNA sequencing method using chemical decomposition method,
A DNA sequencing method characterized by labeling a DNA molecule with a protected amino group, which is then deprotected and bound to a dye molecule after a sequencing reaction. (12) coupling the products of each of the different sequencing reactions with a different colored dye, combining portions of the dye-labeled reactions and electrophoresing them in a polyacrylamide gel;
12. The method according to claim 11, wherein the detection is performed after separation on a gel. (13) A zone containing a raw material for a chromogenically or fluorescently labeled DNA fragment, an electrophoresis gel, a means for introducing the labeled DNA fragment into the zone, and a means for the labeled DNA fragment to move through the gel. A novel apparatus for analyzing DNA fragments, characterized in that it comprises a photometric measuring means for monitoring the analysis of DNA fragments. (14) The novel device according to claim 13, wherein the photometric measuring means is an absorption photometer. (15) The novel device according to claim 13, wherein the light intensity measuring means is a fluorescence photometer. (16) A novel device according to claim 13, in which a DNA fragment is labeled with an amino group and bound to a dye molecule. (17) The novel device according to claim 16, wherein the DNA fragments have different colored labels.