JP2991447B2 - Fluorescent gene identification method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a fluorescent gene identification method.

[Conventional technology]

One of the genetic diagnosis methods is a fingerprint method (Bunseki 1986, No. 7,462-468). After cutting the gene of interest with an enzyme and separating it by agarose gel electrophoresis,
Transfer the pattern to the filter. The filter is immersed in a solution containing a radioactively labeled DNA probe having a specific sequence, and hybridized to a DNA fragment having a sequence complementary to the probe. DNA probe is a ladder
It adheres to a specific band in the DNA fragment group separated in a shape. Attach the film to the filter and remove the DNA
The pattern of the DNA band to which the probe is attached is transcribed.
By selecting the enzyme and DNA probe to be cleaved, a pattern (fingerprint) unique to an individual can be obtained,
Information related to genetic diseases can be obtained. Usually, this fingerprinting method is performed using a ladder pattern obtained by one-dimensional electrophoresis, but it is also performed to increase the amount of information by performing two-dimensional electrophoresis (AGUitterlind
en et.al., Proc. Natl. Acad. Sci. 86 , 2742, (1989)).

On the other hand, attempts have been made to use fingerprints instead of radioactive labels. (Genomics 4, 12
9 (1989)) That is, the color of the fluorescent label is changed for each type of restriction enzyme used for cleavage, and these are simultaneously separated on one migration path and detected in real time.

[Problems to be solved by the invention]

Improving the accuracy of fingerprint diagnosis depends on how many DNA bands can be observed. When one-dimensional electrophoresis is used, if many fragments are cut with multiple enzymes to obtain many bands, bands cannot be separated one by one, resulting in a smear pattern and insufficient information to be obtained. Was. This difficulty is due to the reported real-time fluorescence detection method (Genomi,
cs 4 , 129 (1989) does not help. The separation performance of the device can distinguish between 400 bases and 401 bases when the base sequences match, but even if there is a difference in the number of bases because the difference in mass due to the base type is integrated in a mixture of fragments with different sequences However, it cannot always be said that they can be separated. Separation becomes even more difficult with a fragment having a longer base length, and there is a problem that the fragment cannot be used as a fingerprint for identifying a specimen with high accuracy.

On the other hand, in the two-dimensional electrophoresis method, the number of bands observed is large, but it is necessary to compare the autoradiograms for each sample, which is troublesome and has difficulties in distinguishing between homologues and differences. .

An object of the present invention is to solve these difficulties, obtain highly accurate fingerprint data, and provide an efficient and accurate fluorescent gene identification method.

[Means for solving the problem]

In order to achieve the above object, the fluorescent gene identification method of the present invention cuts a plurality of genes to be distinguished from each other with the same enzyme, and obtains a group of DNA fragments with a different phosphor for each of the plurality of genes. After labeling, these DNA fragment groups were mixed, and the mixture was subjected to enzymatic cleavage of the DNA fragments as a mixture, followed by separation of the DNA fragments as a mixture, and then the separation pattern of the corresponding DNA fragments. Is characterized by optically detecting the difference between the two and distinguishing the plurality of genes comprising the DNA fragments from each other.

As the plurality of genes to which the fluorescent gene identification method of the present invention is applied, genes derived from different individuals for individual identification, normal genes for investigating the cause of genetic diseases, and assumed to be the cause of genetic diseases Gene or a gene derived from an individual.

The DNA fragment separation operation can be performed by one-dimensional or multi-dimensional gel electrophoresis.

Preferred aspects of the apparatus for optically detecting the difference in the separation pattern between the DNA fragments include a mode in which a real-time fluorescence detector capable of line sensing is used, and a mode in which a one-dimensional or two-dimensional gel reader is used. be able to. Further, as a method of optically detecting the difference between the separation patterns of the DNA fragments, by performing optical detection of the DNA fragment group to be detected as a mixture, the same location,
It is extremely important in terms of accuracy and practicality to adopt a method of performing wavelength selection on the fluorescence emitted from each DNA fragment to be compared at the same time.

The combination of the phosphor and the excitation laser light source usable in the present invention includes (FITC and its isomer;
Argon laser 488 nm), (TRITC and Texas Red; argon laser 515 nm or YAG laser 535 nm).

Next, the outline of the fluorescent gene identification method of the present invention is described in the first section.
This will be described with reference to the drawings.

First, the standard sample gene and the sample gene (s)
Is cleaved with an appropriate enzyme (usually an 8-base recognizing enzyme such as NoT-I), DNA fragments obtained from a standard sample gene are labeled with a fluorophore 1 (F1), and DN
The A fragment group is labeled with phosphors 2, ... (F2, ...). in this case,
Adjust with a linker or the like so that the mass numbers of both DNA fragments to be compared are almost the same. Subsequently, a labeled standard sample and a DNA fragment group derived from the specimen are mixed, and the mixture is subjected to enzymatic cleavage of the DNA fragment (using an enzyme such as 4-base recognition) as a mixture, and further, the DNA is obtained by electrophoresis as a mixture. Perform fragmentation operation. In this case, the same sequence of the DNA fragment derived from the standard sample gene and the DNA fragment derived from the sample gene arrives at the detection section almost overlapping. Next, the light emission from the fluorescent substance 1 in the DNA fragment derived from the labeled sample gene and the light emission from the fluorescent substance 2 in the DNA fragment 2 derived from the sample gene are separately received, and the signal intensity is adjusted by adjusting the gain of the light receiving unit amplifier. To be equal. By taking the difference between the signals from both, the DN derived from the standard sample gene
A difference signal generated only when there are mutually different fragments between the A fragment and the DNA fragment derived from the sample gene can be extracted, and the discrimination between the standard sample gene and the sample gene can be performed efficiently and accurately. be able to.

(Operation)

According to the present invention, the labeled DNA fragments derived from a plurality of genes, as a mixture, after cleavage, separation operation, optical detection of the separation pattern difference, it is included in the plurality of genes It is possible to accurately extract the difference spectrum or development pattern difference between labeled DNA fragments, and efficiently identify the differences in the corresponding gene fragment group even when the bands of the electrophoresis pattern of the standard sample or specimen are insufficiently separated. And it can be known with high accuracy.

〔Example〕

Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings. First, a description will be given based on FIG. A normal sample gene was prepared as a standard sample gene, and a genetic disease patient gene was prepared as a sample gene to be tested. These are first cut with the same restriction enzyme NoT-I to obtain respective DNA fragment groups. Next, the absorbance is measured and adjusted so that the mass numbers of both DNA fragments to be compared are approximately the same.

Next, the DNA fragment group derived from the standard sample gene and the DNA fragment group derived from the sample gene are individually fluorescently labeled with the fluorescent substance 1 (F1) and the fluorescent substance 2 (F2), respectively. This fluorescent labeling is performed by binding a fluorescent-labeled nucleotide to the cut end using a complementary chain elongation enzyme using a fluorescent-labeled mononucleotide as a substrate, and binding the fluorescent-labeled nucleotide oligomer to the enzyme cleavage portion by a ligation reaction. May go.

Unreacted fluorescently labeled nucleotides or oligomers using the label are removed by a filter or the like. Measure the fluorescence intensity of the labeled DNA fragment, select the wavelength, and adjust the amount of the sample solution to give almost the same fluorescence intensity when receiving light. Sample 3 by mixing DNA fragment groups
Get.

The DNA fragment is cleaved using the sample 3 as a mixture and using a restriction enzyme Hae III for recognition of 4 bases, etc., under the conditions of a salt concentration of 60 mM to produce fine fragments.

Next, using a mixture 3 consisting of a DNA fragment group derived from the standard sample gene and a DNA fragment group derived from the sample gene obtained by the enzyme cleavage, fluorescence detection was performed by a multicolor photodetection electrophoresis apparatus shown in FIG. Do. That is, first, a mixture 3 composed of the DNA fragment group derived from the standard sample gene and the DNA fragment group derived from the sample gene was injected into a predetermined portion of a 0.3 mm-thick separation gel plate 9 made of polyacrylamide gel using an injector 8. The mixture is electrophoresed, and the F1 labeled standard sample gene derived from the mixture is mixed in the mixture.
The DNA fragment group and the DNA fragment group derived from the sample gene labeled with F2 are separated according to their molecular weights. In this embodiment, the fluorescence detection is performed in real time while performing electrophoretic separation.
An irradiation laser beam 12 emitted from a laser 10 at a position about 25 cm from the starting point of the electrophoresis is reflected by a reflection mirror 11 to irradiate the gel plate from its side. The fluorescent light emitted when the fluorescent-labeled DNA fragment passes through the irradiation light is received by the photodetector 15 constituting a two-dimensional detector or a two-line type line sensor through the image dividing prism 13 and the band-pass filter 14. By passing through the image splitting prism 13, as shown on the monitor 19, the fluorescence image is two lines corresponding to the label F1 of the DNA fragment derived from the standard sample gene and the label F2 of the DNA fragment derived from the sample gene, respectively. It is received as an image.
As described above, by attaching bandpass filters 14 having different transmission wavelengths to the exit surface of the image splitting prism 13,
The light emission from the two types of labels F1 and F2 can be detected separately, and the result is displayed on the display device 18.

As shown in FIG. 1, among the fluorescently labeled DNA fragments derived from the standard sample gene and the sample gene, a fragment common to both does not contribute to the difference spectrum, but a different fragment between the two is a difference spectrum composed of a positive or negative signal. give. This provides information on the differences between the two genes. Furthermore, if you want to know the exact molecular weight using a molecular weight marker, label the molecular weight marker DNA with a fluorescent substance F3 different from F1 and F2, and use the bandpass filter corresponding to F3 with three light exit surfaces of the image splitting prism. Fluorescence can be received and used as a reference signal to determine the molecular weight of the sample. Increasing the number of image divisions can also increase the number of specimens that can be examined simultaneously.

Although the above embodiment is based on one-dimensional electrophoretic separation, the same can be applied to multidimensional electrophoresis.

Further, in the above embodiment, an example was described in which the laser was incident from the gel side as the detection unit and the light was received by the two-dimensional detector.However, the laser was incident from the front, scanning was performed over the irradiation area, and a plurality of Light can be received by a photomultiplier tube.

Instead of real-time measurement, the gel plate may be irradiated after the electrophoresis to measure a two-dimensional image, or data may be obtained by scanning a line sensor with respect to the gel plate.

〔The invention's effect〕

According to the present invention, a plurality of DNA fragments to be distinguished from each other are enzymatically cleaved while being mixed in a mixture, and
By detecting fluorescence and observing the differences in the respective patterns of the corresponding DNA fragments, it is possible to eliminate measurement noise caused by differences in cleavage conditions, electrophoresis conditions, etc., between a plurality of DNA fragments to be identified, and to reduce the nucleotide sequence. Even if they are very similar, it is possible to efficiently and accurately know the difference between the two genes. This method is extremely effective for finding the causative gene site of a genetic disease by comparing a DNA fragment group of a gene common to a patient group with a specific genetic disease with a DNA fragment group of a gene common to a normal person. It is. Further, as another application, a DNA fragment having the same nucleotide length is electrophoresed in a mixed state together with a DNA fragment group of a sample gene using a fragment group having a known nucleotide length as a standard.
Since the A fragments overlap exactly, the DNA length of the sample gene can also be determined accurately.

[Brief description of the drawings]

FIG. 1 is a view for explaining the principle of the method of the present invention, and FIG. 2 is a schematic diagram showing one configuration of an apparatus used for carrying out the method of the present invention. In the figure, 1... A standard gene sample, 2... A sample gene, 3... A mixed sample, 4.
A fragment group, 5: A DNA fragment group derived from an enzyme gene-digested sample gene, 6: A spectrum of a DNA fragment group derived from a standard gene, 7: A spectrum of a DNA fragment group derived from a sample gene,
8 Injector, 9 Separation gel plate, 10 Laser
11: Reflecting mirror, 12: Irradiated laser beam, 13: Image splitting prism, 14: Bandpass filter, 15: Receiver, 16: Controller, 17: Computer, 18: Display device, 19 ……monitor.

Claims (8)

(57) [Claims] Claims: 1. A plurality of genes to be distinguished from each other are cleaved with the same first enzyme, and the obtained DNA fragment group is labeled with a different fluorophore for each of the plurality of genes, and then a fluorescently labeled DN
The A fragment group is mixed, and the DNA fragment is cleaved with the second enzyme in the state of the mixture, and then the DNA fragment is separated in the state of the mixture to obtain a separation pattern of the DNA fragment group derived from the plurality of genes. A fluorescent gene identification method, wherein the plurality of genes are distinguished from each other by optically detecting a difference. 2. The fluorescent gene identification method according to claim 1, wherein said plurality of genes are genes derived from different individuals. 3. The fluorescent gene identification method according to claim 1, wherein the DNA fragment is separated by one-dimensional or multi-dimensional gel electrophoresis. 4. The fluorescent gene identification method according to claim 1, wherein the optical detection of the separation pattern is performed by a real-time fluorescence detector capable of line sensing. 5. The fluorescent gene identification method according to claim 1, wherein the optical detection of the separation pattern is performed by a one-dimensional or two-dimensional gel reader. 6. The fluorescent gene identification method according to claim 4, wherein the optical detection of the separation pattern is performed by wavelength-selecting and detecting fluorescence emitted at the same place and at the same time. 7. A plurality of genes to be distinguished from each other are cleaved with a first enzyme, the obtained DNA fragment groups are labeled with different fluorophores for each of the plurality of genes, and these DNA fragment groups are mixed. Then, a DNA fragment separation operation is performed as a mixture, and then, a difference in separation pattern of the DNA fragment group is optically detected, and the plurality of genes are distinguished from each other by fluorescence. Law. 8. A plurality of genes to be distinguished from each other are cleaved with a first enzyme, and the obtained DNA fragments are labeled with different fluorophores for each of the plurality of genes, and then the fluorescently labeled DNA fragments are collected. Are mixed, and the DNA fragment is cleaved with the second enzyme in the state of the mixture. Then, the DNA fragment is further subjected to electrophoretic separation in the state of the mixture, and the fluorescence intensity of the fluorescence emitted from the phosphor is measured for each fluorescence wavelength. Each DNA separated by electrophoresis
Identifying the electrophoretic distance of the fragments and the gene from which the DNA fragment is derived, detecting a difference in the electrophoretic pattern of a DNA fragment group derived from the plurality of genes, and distinguishing the plurality of genes from each other. Fluorescent gene identification method.