JP3097205B2 - Electrophoretic separation detection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing DNA and RNA, which are genetic materials of humans and other organisms. In particular, the present invention relates to a method that is effective for determining the base sequence of DNA or RNA, or measuring the polymorphism of a restriction enzyme fragment based on the diversity of the base sequence of an individual.

[0002]

2. Description of the Related Art Methods for automatically determining the base sequence of DNA, that is, sequencing using fluorescence detection, are described in Biotechnics, Vol. 5 (1987), pp. 342 to 348 (Bio-Techniques 5 (1987) 342). ~ 34
8). In this method, a fluorescent substance is labeled on a primer used for a DNA sequencing reaction.
At this time, the primers used in the four kinds of reactions corresponding to the four kinds of bases of the DNA are labeled with different fluorophores, respectively, and the generated DNA fragments are separated by electrophoresis into molecular weights in the same migration path. . When the gel, which is the electrophoresis medium, is electrophoresed for a certain distance, the DNA fragment labeled with the fluorescent substance is excited by laser light. Fluorescent substances are identified by detecting the fluorescence emitted at this time and performing wavelength selection,
Obtain the molecular weight separation pattern of the NA fragment. The base sequence of the DNA is determined by analyzing this pattern.

[0003] The molecular weight distribution of restriction fragments of DNA
The method of performing measurement by fluorescence detection is Genomics 4.
Volume (1989), pp. 129-136 (GENOMICS
4(1989) 129-136). this
In the method, cosmid or plasmid DNA is
And cleave the phosphor at the cleavage sites at both ends of the fragment.
The labeled double-stranded DNA oligomer is subjected to a ligation reaction.
As in the method described in the previous paragraph.
Of fluorescent-labeled DNA fragment generated using a simple device
A volume separation pattern has been obtained.

[0004]

SUMMARY OF THE INVENTION By electrophoresis, DN
In the above-described conventional technique in which the nucleotide sequence of A is determined or the molecular weight distribution of the restriction enzyme fragment is measured using fluorescence detection, the wavelength of excitation light cannot be sufficiently separated from the wavelength at which fluorescence is detected. There was a problem that the background fluorescence from the component or the gel was insufficiently reduced, thereby determining the detection sensitivity. Further, since four kinds of phosphors are excited by a single laser beam, there is a problem that the detection sensitivity is determined by the phosphor having the lowest excitation efficiency among the four kinds. Conventionally, it has been substantially impossible to excite four or more types of phosphors with a single laser beam.

SUMMARY OF THE INVENTION An object of the present invention is to provide an electrophoretic separation and detection method for measuring a molecular weight separation pattern of DNA by fluorescence detection in electrophoresis with high sensitivity. To excite various types of DNA
It is to provide a method for identifying and detecting a sample. Another object of the present invention is to provide an electrophoretic separation detection method for measuring a molecular weight separation pattern of DNA with high sensitivity by multicolor detection of fluorescence.
It is to provide a method .

[0006]

According to the present invention, in order to achieve the above object, a nucleic acid fragment sample for which a molecular weight separation pattern is measured by electrophoresis is labeled with at least two kinds of fluorescent substances, and at least one of the fluorescent substances is labeled. By detecting the fluorescence emitted from other types of phosphors by transferring the energy of the fluorescence generated by exciting the phosphor, the molecular weight separation pattern is measured. In addition, two or more types of nucleic acid fragment samples are separated by molecular weight at a place where they have been electrophoresed at a fixed distance in an electrophoresis medium. The nucleic acid fragment sample is identified based on the difference in emission wavelength between the other types of labeled phosphors that emit light due to the transfer of fluorescence energy generated by exciting at least one of the labeled and labeled phosphors. .

[0007] Further, as another means for achieving the above object, two or more kinds of fluorescent substances are labeled on a plurality of types of nucleic acid fragment samples to be identified and detected, and a labeled fluorescent substance is provided for each type of the nucleic acid samples. The distance between them is changed, at least one of the phosphors is excited, and the intensity ratio of fluorescence from the phosphor and another phosphor that emits light by energy transfer is measured.

In particular, for DNA sequencing, a primer which is a single-stranded DNA oligomer used in a sequencing reaction is labeled with two kinds of fluorophores, and a fluorescent label is used to identify the four kinds of bases. The distance between the bodies is set to have four types of distances of 15 bases or less.

[0009]

[Effect] For the efficiency of energy transfer between two kinds of phosphors serving as an energy donor and an energy acceptor, a formula by Von Th. Foerster is known (Anneren der Fusig series)
6.2 (1948) pp. 55-75 (Annalen der
Physik. Folge. Band 2. (1948) pp.55-7
5)). According to this, the energy transfer rate is inversely proportional to the sixth power of the distance between the energy donor and the acceptor, and is proportional to the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. It is also affected by the orientation between the donor and the acceptor.

An experimental study of the distance dependence between energy transfer donors and acceptors has been reported by L. Stryer and RP Haugland (Proceedings of the National Academy of Sciences of Science). USA Volume 58 (196
7 years) pp. 719 to 726 (Proc. Natl. Acad. Sc)
i. USA 58 (1967) 719-726)). This is the result of changing the distance between the donor and the acceptor at both ends by changing the number of peptides of the polypeptide oligomer, and the distance dependence of energy transfer agrees well with Forster's formula. It is shown that. The energy transfer efficiency is 100% when the distance between the donor and the acceptor is 1.2 nm, and 16% when the distance is 4.6 nm.
Has been obtained.

Therefore, single-stranded or double-stranded DNA
If the distance between the labeled energy donor and the acceptor phosphor is several nm or less and the overlap between the emission spectrum of the former and the absorption spectrum of the latter is large, energy transfer will occur between the two, Moreover, 10% or more 1
Energy transfer with an efficiency close to 00% can be expected. That is, when the phosphor serving as the energy donor is excited, highly efficient light emission from the energy acceptor can be observed. Also,
Compared to the case where only the fluorescent substance serving as the energy donor is labeled, the emission wavelength shifts to the longer wavelength side, so that the wavelength of the excitation light is separated from the wavelength of the emission light. For this reason, the measurement is less susceptible to scattering of excitation light and the like. For the above reasons, highly sensitive fluorescence measurement can be realized.

When a plurality of light emitters capable of being excited at the same wavelength are selected, if light emission by energy transfer is used, a plurality of acceptors receiving energy transfer with high efficiency to the same energy donor can be selected. Combination, or a combination of multiple phosphors that can be excited with high efficiency at the same wavelength and a receptor that receives energy transfer from each with high efficiency, etc., can be excited with high efficiency.
That is, the range of choice of the fluorescent substance that can be detected with high sensitivity is expanded.

The case of energy transfer between two molecules between an energy donor and an acceptor has been described above. However, the shift between the excitation wavelength and the emission wavelength can be increased, and the number of illuminants that can be simultaneously detected can be increased. To do this, energy transfer between three or more molecules can be utilized. That is, first, the phosphor on the shortest wavelength side is excited, and energy is transferred between the phosphor and a second phosphor having an excitation spectrum that overlaps greatly to excite the phosphor. Next, energy transfer occurs between the fluorescence spectrum of the second phosphor and the third phosphor having an excitation spectrum having a large overlap, thereby exciting the third phosphor. By sequentially exciting the phosphors having the fluorescence spectrum on the longer wavelength side in this manner, it becomes possible to increase the shift between the excitation wavelength and the emission wavelength, and to increase the number of illuminants that can be simultaneously detected.

On the other hand, when the distance between the energy donor and the acceptor is changed, the efficiency of energy transfer also changes. That is, by changing the distance between the energy donor and the acceptor, the intensity ratio between the light emission from the energy donor and the light emission from the acceptor can be changed.
Therefore, by measuring the intensity ratio of the luminescence from the energy donor to the luminescence from the acceptor by changing the distance for each type of nucleic acid sample with the same combination of the energy donor and the acceptor, The sample can be identified.

If the efficiency of energy transfer is about 10% or more, sufficient light emission can be obtained as compared with a case where four kinds of phosphors are excited by a single laser. Therefore, the distance between the energy donor and the acceptor may be about 15 bases or less for both the single-stranded DNA and the double-stranded DNA. That is, in a double-stranded state, the distance between base pairs is 1
The average distance is several nm or less in consideration of the folding of the chain even in a single-stranded state, taking into account several nm in 5 bases.

In the case of DNA sequencing, by changing the distance between the energy donor and the acceptor for each of the four types of primers used for the sequencing reaction, ie, for each type of single-stranded DNA oligomer, The base species of DNA fragments of various lengths, which are the products of the sequence reaction, can be identified.

[0017]

【Example】

First Embodiment Hereinafter, a first embodiment according to the present invention will be described with reference to FIGS. In this example, using λ phage DNA as a model substance, samples obtained by digesting the λ phage DNA with different combinations of restriction enzymes were labeled with different combinations of fluorophores, and then electrophoresed simultaneously on the same migration path. Differences in patterns are identified by differences in emission wavelength.

In this embodiment, double-stranded DNA oligomers 1a and 1b labeled with two kinds of fluorescent substances which generate energy transfer between them are introduced into a cleavage site of a restriction enzyme HindIII by a ligation reaction. As shown in FIG. 1, fluorescein isothiocyanate (FITC) 2 which can be efficiently excited by an argon ion laser (emission wavelength: 488 nm) is used as an energy donor. As an energy acceptor, tetramethylrhodamine isothiocyanate (TRITC) 3, whose sulfo spectrum has a large overlap with the fluorescence spectrum of FITC2, and sulfolodamine 10
1 sulfonic acid derivative (Texas Red)
4 is used.

The double-stranded DNA oligomer used for the introduction of the labeled fluorescent substance by the ligation reaction has the following structure: 5'-CGTTGTAAAACGACCGCCAGT-3 '3'-GCAACATTTTGCTCGCCGGTCATCGA-5' It conforms to the shape and selectively binds to the cleavage site of the HindIII cleavage fragment by a ligation reaction.

The label position of the fluorescent substance is as follows: FITC2 is the phosphorus portion between the twelfth C and the thirteenth T from the 3 'side of the lower chain, and TRITC3 and Texas Red 4 are 5' of the upper chain. And the phosphorus portion between the third T and the fourth T. The method disclosed in JP-A-61-44353 is used for labeling the oligonucleotide of the energy donor and the acceptor. In this method, a phosphoric acid bond at a phosphor-labeled site of a polynucleotide is replaced with a phosphonic acid bond having a functional group, and the functional group is combined with the phosphor to realize phosphor labeling. This is a technique that allows the introduction of a label to the target.

In this embodiment, the case where the phosphor label is bonded to the phosphorus atom of the nucleotide skeleton has been described. However, the label position is not limited to this.
8 (1987) 336-341 (Science, 2
38 (1987) pp. 336-341), and it is also possible to label the base.

The double-stranded oligomer is prepared by separately synthesizing each of the constituent chains, and thereafter annealing to form a double-stranded chain.

Fluorescently labeled double-stranded DNA oligomer 1
FIG. 2 shows the respective fluorescence spectra of a and 1b. This is a spectrum when excited at 488 nm, which is the oscillation wavelength of an argon laser, and a sufficient amount of energy transfer occurs between each energy donor and acceptor. Moreover, an emission intensity equal to or higher than that obtained when excited by a He-Ne laser having an oscillation wavelength of 543 nm that can excite TRITC3 and Texas Red 4 in common can be obtained. Therefore, each laser (argon laser, 〜１０10 mW,
Considering the intensity of the excitation light obtained with a He-Ne laser (up to 1 mW) and the intensity of the scattered light due to the difference between the excitation wavelength and the emission wavelength, more sensitive fluorescence detection is realized by using energy transfer. You can do it.

The preparation of a DNA sample is performed in the following procedure. λ
To a degradation product (λ / HindIII; commercially available product) 5 obtained by digesting the phage DNA with the restriction enzyme HindIII, about 10 times the amount of each of the fluorescently labeled double-stranded DNA oligomers 1a and 1b was added,
Ligation reaction 6 is performed at 16 ° C. for 30 minutes using a commercially available ligation kit, and the reaction product is purified by ethanol precipitation. Next, the reaction product containing Texas Red 4 was added to 50 mM Tris-HCl (pH 7.5), 1
0 mM MgCl ₂ , 1 mM Dithiothreitol, 50 mM
Restriction enzyme in buffer at 37 ° C. consisting of NaCl
The restriction enzyme digestion reaction 7 is performed with HinfI, and the reaction product is purified by ethanol precipitation.

Next, these reaction products are mixed 8, injected into the same electrophoresis path, and subjected to electrophoresis 9. Glass plate 6
3% acrylamide gel is used as the gel 60 for electrophoresis sandwiched between 1. A voltage of about 30 V / cm is applied between the upper end and the lower end of the gel 60. The fluorescence is measured by causing an argon laser beam 63 from a laser light source 62 to be incident perpendicularly to the migration direction of the DNA fragment 70 from the end face of the gel 60 by a reflecting mirror 64 at a migration distance of 10 cm. An emission image generated when each DNA fragment 70 in the reaction product passes through the laser light irradiation site is received by the fluorescent two-color detection device. The fluorescent two-color detection device includes a prism 65 that divides a linear image of a laser beam irradiation part into two, an optical filter 66 through which a beam from each image passes, and a lens system that forms an image. 6
7 and a two-dimensional detector 68. The optical filter 66 has a center wavelength of 580 nm, which mainly transmits light emitted from TRITC3 and a half-value width of 40 nm, and a central wavelength 615, which mainly transmits light emitted from Texas Red 4.
The half-width of 30 nm was used. Two-dimensional detector 6
8 is a CCD with an image intensifier
A camera was used. The signal from the two-dimensional detector 68 is processed by the data processing device 69, and the light emission from each phosphor is identified by color correction calculation. In the color correction calculation, the emission intensity of each phosphor is obtained by a linear combination of the emission intensity values of the two types of emission spectra observed, based on the parameters obtained from the respective emission spectra and the transmission characteristics of the optical filter. FIG. 3 shows an example of the observed signal. The signal from the fragment obtained by digesting the phage DNA with HindIII and the signal from the fragment further digested with Hinfl can be clearly distinguished and detected.

In this example, λ phage DNA was used as a model sample, but this technique is effective for the following uses. First, a certain restriction enzyme digest of DNA whose molecular weight distribution of the restriction enzyme fragment is to be investigated and a specific restriction enzyme digest of which the molecular weight distribution of the restriction fragment is known are separated into different fluorescent substances. Labeling is performed in combination, and comparison of each electrophoresis pattern is performed by identifying each emission wavelength. This makes it possible to determine the molecular weight distribution of the DNA fragment. As another example, for example, genomic DNA of bacteria of different strains
Are labeled with different combinations of phosphors,
The comparison of the electrophoretic patterns of the digested fragments by the same combination of the same restriction enzymes is performed. By discriminating the emission wavelengths of the respective fluorophores and determining the difference, only the difference between the DNA digested fragments due to the difference in the strains Can be selectively detected. The latter example is also effective when comparing individuals with a genetic disease and normal individuals.

Second Embodiment A second embodiment according to the present invention will be described with reference to FIGS. The present embodiment is directed to the determination of the base sequence of DNA. The DNA sequencing reaction is based on the Sanger method. A combination of the following four types of fluorophores is used as sequencing primers 33, 34, 35, 36 corresponding to the four types of bases of DNA. The first two are the same combination as the first embodiment, FITC28 and TRITC2
9, and Texas Red 30, and as the remaining two, a combination of a succinylfluorescein derivative (SF526) 31 having an emission peak at FITC 28 and 526 nm and a phthalocyanine derivative 32 having an emission peak at FITC 28 and 680 nm was used. . FITC
28 and each of the above phosphors are labeled on the primer with a distance of about 4 bases. The labeling of the energy donor and the acceptor with the oligonucleotide was performed in the same manner as in the first example.
The method disclosed in JP-A-61-44353 is used. The following labeling method is used to label the energy donor and acceptor one by one on the oligonucleotide. First, the oligonucleotide having a phosphonic acid introduced at the above-mentioned label position is labeled with F
A reaction for labeling ITC28 is performed. At this time, the reaction product was monitored at any time by liquid chromatography, and the absorbance at 260 nm derived from DNA and 490 nm derived from FITC28 were monitored.
By comparing the absorbances of the above, the reaction is stopped at a stage where FITC28 has been introduced to some extent and not so many FITC28s have been introduced. The reaction product is subjected to electrophoresis in an acrylamide gel to separate an unlabeled oligonucleotide from one into which one and two FITC28s have been introduced. The oligonucleotide to be purified is purified. The FITC-labeled DNA oligomer thus produced is subjected to a reaction for labeling TRITC 29, Texas Red 30, SF526 31 and phthalocyanine derivative 32, respectively. By the above reaction process, TRITC29, Texas Red 3
Thus, DNA oligomers 33, 34, 35 and 36 in which one of 0, SF52631 and phthalocyanine derivative 32 and FITC 28 are labeled one by one are obtained. The saxinylfluorescein derivative and the phthalocyanine derivative are disclosed in JP-A-1-180455 and PCT application WO8, respectively.
It can be synthesized by the method disclosed in JP-A-8 / 04777.

The sequence reaction 37 is performed according to a usual reaction protocol. Reaction products 38, 39,
As shown in FIG. 5, a mixture 8 of 40 and 41 is sandwiched between glass plates 61, and electrophoresis 9 is carried out on the same migration path of acrylamide gel 60 mixed with urea under denaturing conditions.
The fragment A is separated according to its molecular weight, and a laser beam 47 is incident from the side of the gel 60 perpendicular to the migration path, so that the DNA
The fluorescence emitted from the fragments is detected. The measurement 44 of the light emission from the labeled fluorescent material on the DNA fragment is detected by the same combination of the excitation laser light, the electrophoresis apparatus, and the photodetector as in the first embodiment. The difference from the first embodiment lies in the image division portion of the light emission image shown in FIG. 5. In this embodiment, in order to detect four kinds of light emission, a pentagonal prism 48 and four types having different transmission characteristics are used. The image 52 is formed on the image forming surface 51 by the image forming lens system 50 using the optical filter 49 described above.

In this embodiment, as an alternative to the fourth probe, as shown in FIG. 6, three kinds of fluorescent substances, FITC28, Texas Red 30, and phthalocyanine derivative 32, are separated by about 4 bases each to label the primer. And the T reaction primer 36, and the luminescence from the phthalocyanine derivative 32 can be observed with high efficiency.

In this example, energy transfer between three molecules is used. That is, first, the phosphor FITC28 having the emission peak on the shortest wavelength side is excited, and energy transfer is performed between the second phosphor, Texas Red 30, which has an excitation spectrum that overlaps greatly with the fluorescence spectrum of this phosphor. Wake up and excite this. Next, a third phosphor, a phthalocyanine derivative 32, having an excitation spectrum that overlaps greatly with the fluorescence spectrum of the second phosphor
Energy transfer occurs between and excites this.
By sequentially exciting the phosphors having the fluorescence spectrum on the long wavelength side in this manner, the shift between the excitation wavelength and the emission wavelength is increased.

Third Embodiment Hereinafter, a third embodiment according to the present invention will be described with reference to FIG. The present embodiment is directed to the determination of the base sequence of DNA. The DNA sequencing reaction is based on the Sanger method. In this example, the following primers are used as sequencing primers. As a combination of phosphors, an argon ion laser (oscillation wavelength 4
Fluorescein isothiocyanate (FITC) 71, which can be efficiently excited at 88 nm), as an energy acceptor, the excitation spectrum largely overlaps with the fluorescence spectrum of FITC 71, and the maximum of the fluorescence spectrum is FITC 71.
And a sulfonated chloride derivative (trade name: Texas Red) 72 of sulfolodamine 101 which is separated from that of the above by nearly 100 nm. Four types of primers 73, 74, 7
FIT on primers corresponding to 5,76
Change the distance between C71 and Texas Red 72. That is, FITC71 and Texas Red 72 are labeled at a distance of 2, 5, 9, or 12 bases. The nucleotide sequence of the primer was 30 mer immediately upstream of the cutting site of the M13 phage with the restriction enzyme.

The labeling of the oligonucleotide with the energy donor and the acceptor was carried out in the same manner as in the first embodiment.
The method disclosed in JP-A-61-44353 is used.
The following labeling method is used to label the energy donor and acceptor one by one on the oligonucleotide. First, a reaction of labeling FITC71 with the oligonucleotide having a phosphonic acid introduced at the label position is performed. At this time, the reaction product is monitored at any time by liquid chromatography, and DN
Absorbance at 260 nm from A and 49 from FITC71
By comparing the absorbance at 0 nm, the reaction is stopped at a stage where FITC71 has been introduced to some extent and not so many FITC71s have been introduced. The reaction product is subjected to electrophoresis in an acrylamide gel to separate an unlabeled oligonucleotide from one into which one and two FITC71 have been introduced, cut out a gel containing a portion into which one FITC71 has been introduced, and contained the gel therein. The oligonucleotide to be purified is purified. A reaction for labeling Texas Red 72 with the FITC-labeled DNA oligomer thus generated is performed. Through the above reaction process, DNA oligomers 73, 74, 75 and 76 in which one FITC 71 and one Texas Red 72 are labeled are obtained.

The DNA oligomer prepared as described above,
That is, a sequence reaction 37 is performed for each base type using the primers 73, 74, 75, and 76. Next, these reaction products 38, 39, 40, and 41 are mixed 8 and subjected to electrophoresis 9 in the same migration path. As the gel 60 for electrophoresis, a 4.5% acrylamide gel sandwiched between glass plates 61 and containing urea as a denaturant is used. A voltage of about 30 V / cm is applied between the upper end and the lower end of the gel 60. Fluorescence was measured at gel distance of 30 cm.
Argon laser light 6 from the laser light source 62 from the end face
3 is performed by using a reflecting mirror 64 and making the light incident perpendicularly to the migration direction. An emission image generated when each DNA fragment 70 in the reaction product passes through the laser light irradiation site is received by the fluorescent two-color detection device. The fluorescence two-color detection device is the same as that shown in FIG. 1 described in the first embodiment. The optical filter 66 has a central wavelength of 520 nm for transmitting light emitted from the FITC 71 and a half-value width of 30 nm, and a central wavelength 61 for transmitting light emitted from Texas Red 72.
5 nm and a half value width of 30 nm were used.

When these DNA fragments for each base type are excited by an argon laser having an oscillation wavelength of 488 nm, FITC7
Since the efficiency of energy transfer differs according to the distance between Texas Red 1 and Texas Red 72, FI having a peak at 520 nm
The four types of primers 38, 39, 40, and 41 can be identified by measuring the intensity ratio of the emission of TC71 and the emission of Texas Red 72 having a peak at 620 nm. That is, the signal from the two-dimensional detector 68 is processed by the data processing device 69, the light emission from each phosphor is identified, the intensity ratio is calculated, and the base type is specified.

In this embodiment, the sequencing by electrophoresis using the flat gel 60 has been described, but the present invention is not limited to this. In particular, from 50 μm to 1
In electrophoresis in a gel or electrolyte solution in a single capillary tube of about 00 μm, ie, sequencing by capillary electrophoresis, it is essential to simultaneously migrate four types of samples for each base type, so that the effect is large.

In the present embodiment, FITC 71 and Texas Red 72 which can be excited by an argon ion laser as a combination of phosphors which cause energy transfer therebetween.
However, the present invention is not limited to this. In particular, by selecting an appropriate combination of phthalocyanine derivatives capable of changing its fluorescence spectrum in the region from 650 nm to 750 nm by changing the functional group or the central metal, it becomes possible to excite with a semiconductor laser. In addition, the size and cost of the device can be reduced.

Fourth Embodiment A fourth embodiment according to the present invention will be described with reference to FIG. In the present embodiment, the simultaneous sequence of the sense strand and the antisense strand of DNA in the same migration path is realized. In this example, the DNA inserted into the plasmid PUC18 was used.
Target the sample. Primer for sense strand 87,8
8, 89 and 90 are succinylfluorescein derivatives (SF526) 83 which can be efficiently excited by an argon ion laser as an energy donor and whose emission spectrum has an emission peak at 526 nm which largely overlaps the excitation spectrum of Texas Red 84. , Labeled with Texas Red 84 as an energy acceptor. The distance between the energy donor and the acceptor in the above four primers is 2 bases, 5 bases, 9 bases and 12 bases as in the case of the third embodiment. Sequencing primer in the opposite direction, antisense strand direction primer 91,
92, 93, and 94 can also be efficiently excited by an argon ion laser and serve as SF5 as energy donors.
50, clearly distinguishable from the 26 83 fluorescence spectrum
A succinylfluorescein derivative (SF505) 85 having an emission peak at 5 nm, as an energy acceptor, the excitation spectrum of which overlaps greatly with the fluorescence spectrum of SF505 85, and the maximum of the fluorescence spectrum is 580 nm and that of SF505 85, SF526 83 and Texas Red 84. Labeled with clearly distinguishable tetramethylrhodamine isothiocyanate (TRITC) 86. The distance between the energy donor and the acceptor in the above four primers was the same as that used in the third embodiment.
They are 2, 5, 9 and 12 bases. The base sequence is 30 bases immediately downstream of the cutting site of PUC. Note that SF505 and SF526 are disclosed in
It can be synthesized by the method disclosed in -180455.

The sequence reactions 95 and 6 are carried out according to a usual reaction protocol. Reaction product 97a for each base species,
97b was mixed 8 and, as shown in FIG.
In the same electrophoresis path in the acrylamide gel 60 under denaturing conditions in which urea is mixed,
DNA fragments are separated by their molecular weight. The measurement 44 of the emission of the fluorescent substance based on the energy transfer from the fluorescent label on the DNA fragment is performed by the same combination of the laser beam for excitation, the electrophoresis apparatus, and the photodetector as shown in FIG. To detect. The difference from the first embodiment is the image division portion of the emission image shown in FIG. 5. In this embodiment, a pentagonal prism 48 and a pentagonal prism 48 are used to detect four types of emission excited by the laser beam 47. Four types of optical filters 49 having different characteristics are used. With the above optical system, it is possible to identify and detect light emission from four types of phosphors. SF526 83 and Texas Red 8 at data processing device 69
By calculating the emission intensity ratio between 4, SF505 85 and TRITC26, the direction of the DNA to be analyzed and the sequence of the base type can be specified.

Fifth Embodiment A fifth embodiment according to the present invention will be described with reference to FIG. In this example, using λ phage DNA as a model substance, samples obtained by digesting the λ phage DNA with different combinations of restriction enzymes were labeled with energy donors and acceptors having different distances from each other, and were labeled on the same migration path. Simultaneous electrophoresis is performed, and the difference in the pattern is identified based on the difference in the emission intensity ratio between the donor and the acceptor.

In this example, double-stranded DNA oligomers 1c and 1d labeled with two kinds of fluorescent substances which generate energy transfer therebetween are introduced into the cleavage site of the restriction enzyme HindIII by ligation reaction 6. FITC is used as an energy donor, and Texas Red 4a, 4b is used as an energy acceptor. The DNA double-stranded oligomer used to introduce the labeled fluorescent substance by the ligation reaction 6 has the following structure: 5′-CGTTGTAAAACGACGGCCAGT-3 ′ 3′-GCAACATTTTTGCTGCCGGTCATCGA-5 ′ The ligation reaction 6 selectively binds to the cleavage site of the HindIII cleavage fragment.

Regarding the labeling position of the fluorescent substance, FITC2 is located at the phosphorus portion between the third T and the fourth T from the 5 'side of the upper chain, and Texas Red 4a and 4b are respectively located at the 3rd position of the lower chain. The phosphorus portion between the 7th T and the 8th T from the 3 'side, and the phosphorus portion between the 12th C and the 13th T from the 3' side, and the energy donor and acceptor oligos. A method similar to that of the third embodiment is used for labeling nucleotides. For the double-stranded oligomer, each of the constituent chains is separately synthesized, and later converted into a double-stranded chain by annealing.

The preparation of a DNA sample is performed according to the following procedure. λ
To a degradation product (λ / HindIII; commercially available product) 5 obtained by digesting the phage DNA with the restriction enzyme HindIII, about 10 times the amount of each of the fluorescently labeled double-stranded DNA oligomers 1c and 1d was added,
Ligation reaction 6 is performed at 16 ° C. for 30 minutes using a commercially available ligation kit, and the reaction product is purified by ethanol precipitation. Next, a reaction product containing Texas Red 4b between the twelfth C and the thirteenth T from the 3 'side was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl ₂ , 1 mM Dithiothreitol, 50 mM N
Restriction enzyme Hinf in a buffer consisting of aCl at 37 ° C
The restriction enzyme digestion reaction 7 is performed by I, and the reaction product is purified by ethanol precipitation.

Next, these reaction products are mixed 8 and subjected to electrophoresis 9 on the same electrophoresis path under the following conditions. As the gel for electrophoresis 9, a 3% acrylamide gel is used. Apply a voltage of about 30 V / cm between the upper and lower ends of the gel,
The measurement 44 of the light emission is performed by injecting argon laser light perpendicularly to the migration direction from the end surface of the gel at a migration distance of 10 cm. An emission image generated when each DNA fragment 70 in the reaction product passes through the laser light irradiation site is detected by a fluorescent two-color detection device as shown in FIG. The same two-color fluorescence detector as that of the third embodiment is used. By analyzing data of the luminescence intensity around 520 nm and the luminescence intensity around 620 nm, the signal from the fragment obtained by digesting the λ phage DNA with HindIII and the signal from the fragment obtained by digesting the λ phage DNA with HinfI are clearly identified and detected. be able to.

In this example, λ phage DNA was used as a model sample, but this technique is effective for the following uses. First, a certain restriction enzyme digest of DNA whose molecular weight distribution of the restriction enzyme fragment is to be investigated and a specific restriction enzyme digest of which the molecular weight distribution of the restriction fragment is known are separated from each other by a distance from each other. Labeling is performed with a combination of different phosphors, and the comparison of each electrophoresis pattern is performed by identifying the emission intensity of each phosphor. Thereby, the molecular weight distribution of the DNA fragment can be obtained. As another example, for example, genomic DNAs of different bacterial strains are labeled with a combination of fluorophores having different distances from each other, and a comparison of electrophoresis patterns of digested fragments by the same combination of the same restriction enzymes is performed.
By discriminating the respective luminescence intensities and determining the difference, it is possible to selectively detect only the difference portion of the DNA digestion fragment due to the difference of the strain. The latter example is also effective when comparing individuals with a genetic disease and normal individuals.

[0045]

According to the present invention, fluorescence can be detected in the same electrophoresis path in electrophoresis by sufficiently separating the wavelength of the excitation light and the wavelength of the emitted light. Can be realized. In addition, since various kinds of phosphors can be excited with a single laser beam with high efficiency, it is possible to realize a highly sensitive and large-throughput measurement of a DNA molecular weight separation pattern.

Further, according to the present invention, discrimination of four or more DNA samples can be realized by two kinds of phosphors (dyes), and a large number of DNA samples can be simultaneously discriminated by a single laser beam on the same migration path. And can be detected. In addition, more sensitive detection can be realized than in the case where the fluorescent substance is changed for each type of DNA sample. Therefore, it is possible to greatly improve the throughput of DNA sequencing. For example, simultaneous sequencing of four or more DNA samples, simultaneous sequencing of sense and antisense strands, and the like are possible. Furthermore, simultaneous measurement of the molecular weight distribution of restriction enzyme cleavage fragments of four or more DNA samples can be realized. In particular, in the case of sequencing using capillary electrophoresis, it is essential to identify four types of samples for each base type, so that the effect of the present invention is great.

[Brief description of the drawings]

FIG. 1 is a schematic view showing a first embodiment according to the present invention.

FIG. 2 is a graph showing characteristics of components in the first embodiment according to the present invention.

FIG. 3 is a graph showing an example of a result in the first example according to the present invention.

FIG. 4 is a schematic diagram showing a second embodiment according to the present invention.

FIG. 5 is a configuration diagram of a detection unit according to a second embodiment of the present invention.

FIG. 6 shows an alternative example of the primer for T reaction in the second embodiment according to the present invention.

FIG. 7 is a schematic diagram showing a third embodiment according to the present invention.

FIG. 8 is a schematic diagram showing a fourth embodiment according to the present invention.

FIG. 9 is a schematic view showing a fifth embodiment according to the present invention.

[Explanation of symbols]

1a, 1b, 1c, 1d: fluorescently labeled double-stranded oligomer, 2, 28, 71: FITC, 3, 29, 86 ... T
RITC, 4, 4a, 4b, 30, 72, 84 Texas red, 5 λ / HindIII, 6 ligation reaction, 7 restriction enzyme digestion, 8 mixing, 9 electrophoresis,
19: spectrum when the receptor is TRITC, 20: spectrum when the receptor is Texas Red, 21: FIT
C peak, 22 ... TRITC peak, 23 ... Texas red peak, 24 ... 580 nm signal, 25 ... 6
15 nm signal, 26 ... TRITC signal, 27 ... Texas Red signal, 31,83 ... SF526, 32 ... phthalocyanine derivative, 33, 73 ... primer for A reaction, 34, 74 ... primer for C reaction, 35, 75 ... G
Reaction primer, 36,76 ... T reaction primer,
37 ... sequence reaction, 38 ... reaction product, 39 ... C
Reaction product, 40: G reaction product, 41: T reaction product, 44: measurement of light emission, 47: laser beam, 48: pentagonal prism, 49: optical filter, 50: imaging lens system, 51: imaging Surface, 52 image, 60 gel, 61 glass plate, 62 laser light source, 63 laser light, 64 reflecting mirror, 65 prism, 66 optical filter, 67 imaging lens system, 68 two-dimensional Detector, 69: Data processing device, 70: DNA fragment, 85: SF505, 87, 8
8, 89, 90: sense strand direction primer, 91, 9
2, 93, 94: antisense strand primer, 95
... sense strand direction sequence reaction, 96 ... antisense strand direction reaction, 97a, 97b ... sequence reaction product.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuko Kawamoto 1-280 Higashi Koikekubo, Kokubunji-shi, Tokyo Hitachi Central Research Laboratory, Inc. (72) Inventor Satoshi Takahashi 1-280 Higashi Koikekubo, Kokubunji-shi, Tokyo Hitachi, Ltd. Central Research Laboratory (72) Inventor Tetsuo Nishikawa 1-280 Higashi Koikekubo, Kokubunji-shi, Tokyo Hitachi, Ltd. Central Research Laboratory Hitachi, Ltd. (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 21/78 C12Q 1/68 G01N 27/477 G01N 33/58

Claims (17)

(57) [Claims] 1. A method for electrophoresing a nucleic acid fragment sample to which a fluorescent substance is bound.
To detect the fluorescence emitted from the fluorophore and cut the nucleic acid.
In the electrophoretic separation detection method for detecting a piece sample, the method
Of the multiple types of fluorophores bound to nucleic acid fragment samples,
Energy from at least one kind of excited phosphor
The fluorescence emitted from the other types of phosphors
An electrophoretic separation and detection method characterized by detecting. 2. A method for electrophoresing a nucleic acid fragment sample to which a fluorescent substance is bound.
To detect the fluorescence emitted from the fluorophore and cut the nucleic acid.
In the electrophoretic separation detection method for detecting one sample,
A plurality of types respectively bound to the types of nucleic acid fragment samples
At least one kind of the excited fluorescence of
Due to the transfer of energy from the body, other types of said fluorescence
The nucleic acid fragment test is performed according to the emission wavelength of fluorescence emitted from the body.
Electrophoretic separation test characterized by identifying the type of material
How to get out. 3. The method for detecting and separating electrophoresis according to claim 2,
In this case, a plurality of kinds of the above-mentioned phosphors are on the same strand of a DNA strand.
An electrophoretic separation and detection method comprising binding. 4. The method for detecting and separating electrophoresis according to claim 3,
The phosphor is of two types, and the phosphor is D
Electrophoretic components characterized by binding to the same NA chain
Separation detection method. 5. The method for detecting and separating electrophoresis according to claim 2,
In the above, a plurality of types of the above-mentioned phosphors are used for each of DNA double strands.
An electrophoretic separation detection method comprising binding to a strand. 6. The method for detecting and separating electrophoresis according to claim 5,
In the above, there are two types of the phosphor, and the phosphor is
Characterized by binding to each strand of the DNA duplex.
Electrophoretic separation detection method. 7. A plurality of kinds of phosphors are bound, and said phosphors
Energy from at least one of the excited phosphors
The movement of the energy excites the other types of phosphors to generate fluorescence.
Characterized by being used in a sequence reaction
Primer. 8. A first phosphor and said excited first phosphor.
Excited by the transfer of energy from an optical body to emit fluorescence
Is combined with the second phosphor and used for the sequencing reaction.
Primer. 9. The primer according to claim 8, wherein
The distance between the first phosphor and the second phosphor is 15
A primer characterized by having no more than a base. 10. The primer according to claim 8, wherein
The distance between the first phosphor and the second phosphor is 4
A primer, which is a base. 11. A first phosphor and the excited first phosphor
Excited by transfer of energy from the phosphor to emit fluorescence
Use a primer that binds to the second phosphor
A method for determining a nucleotide sequence of DNA, characterized by the following. 12. The nucleotide sequencing of the DNA according to claim 11.
In the method, the type of the second phosphor is different.
Primers are used for each sequencing reaction with different terminal bases.
A method for determining a nucleotide sequence of DNA, which is used. Claim 13 The nucleotide sequencing of the DNA according to claim 11.
The first phosphor and the second phosphor
The primer having a different distance between
It is characterized in that it is used for each sequence reaction with different base species.
DNA sequencing method. 14. Energy donor and energy acceptor
Electrophores the nucleic acid fragment sample to which
The nucleic acid fragment sample thus separated is irradiated with light to be excited.
Said Transfer of energy from the energy donor
Detects fluorescence emitted from the excited energy receptor
A method for detecting and separating electrophoresis. 15. Energy supply excited by light irradiation
Donor and the energy from the excited energy donor
Energy transfer to the fluorescent energy receptor
A nucleic acid fragment sample to be combined, comprising:
Different energy acceptors are bound for each type of end base
The separated nucleic acid fragment sample is separated by electrophoresis and separated.
Irradiating the nucleic acid fragment sample with the light
Electricity characterized by detecting fluorescence emitted from a receptor
Electrophoretic separation detection method. 16. Energy supply excited by light irradiation
Donor and the energy from the excited energy donor
Energy transfer to the fluorescent energy receptor
Combined and used for sequence reactions
Primer. 17. Energy supply excited by light irradiation
Donor and the energy from the excited energy donor
Energy transfer to the fluorescent energy receptor
A nucleic acid fragment sample to be combined, comprising:
Different energy acceptors are bound for each type of end base
The separated nucleic acid fragment sample is separated by electrophoresis and separated.
Irradiating the nucleic acid fragment sample with the light
The nucleic acid fragment sample according to the wavelength of fluorescence emitted from the receptor
And electrophoretic separation detection.