JPH0330700A - Detection of nucleic aid region - Google Patents

Abstract

PURPOSE:To simultaneously and readily detect plural nucleic acid regions by amplifying the plural regions having mutually different size in a sample gene with a gene-amplifying method, subjecting the sample to electrophoresis, separating the amplified plural regions according to the size of individual region and detected nucleic acid fragments. CONSTITUTION:Plural regions to be detected having mutually different size in sample DNA or RNA are dissolved in buffer solutions and subjected to heat treatment to denaturate DNA or RNA protease. Then nucleotide and DNA or RNA polymerase are added, thus subjected to a temperature-controlling in a temperature cycle of high temperature and low temperature by a gene- amplifying method to simultaneously amplify the plural regions to be detected. Next, the sample after the amplification is subjected to electrophoresis and separated according to the respective sizes of the amplified plural regions to be detected, then a separated nucleic acid region is detected by detecting a nucleic acid fragment.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for detecting a nucleic acid region. More specifically, the present invention relates to a method for detecting a nucleic acid region that can simultaneously detect multiple regions to be detected in a DNA or RNA sample. The present invention is based on gene analysis (applicable to diagnosis of various genetic diseases and infectious diseases, quality check of vaccines and biological products, etc.).

[Prior art 1] Conventionally, whether or not a DNA or RNA (hereinafter sometimes referred to as "nucleic acid") sample has a specific region has been investigated by Southern blot method, Northern blot method, dot plot method, etc. was.

Recently, a gene amplification method that can detect even one copy of a DNA region has been developed (R.

5aiki et al., 5science, 230.1350 [
1985)), the gene amplification method uses two types of primers and DNA or RNA polymerase that hybridize to both ends of the nucleic acid region to be detected, and cycles of complementary strand synthesis and denaturation are repeated several thousand times to achieve detection. Amplify the target area hundreds of thousands of times. Therefore, it is possible to detect even a very small amount of a nucleic acid region in a sample, and it is also possible to detect one copy of a nucleic acid region in a sample.

Gene analysis methods using gene amplification methods are also under scrutiny; for example, Journal of C1inic
alLaboratory Analysis 2:1
94-198 (19881) describes a method for detecting the provirus of human T-cell leukemia virus using gene amplification methods.

However, conventional detection methods using gene amplification methods can only detect one region in one operation. Therefore, when it is necessary to detect multiple regions in a nucleic acid, detection operations (amplification by gene amplification method and hybridization with a probe) must be performed for each region, which is time-consuming and labor-intensive. Met.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a method for detecting a nucleic acid region that can easily detect a plurality of nucleic acid regions simultaneously.

[Means for Solving the Problems] As a result of intensive research, the inventors of the present application have made the sizes of the nucleic acid regions to be detected and amplified by the gene amplification method different, and combined the gene amplification method and electrophoresis. We have completed the present invention by discovering that multiple nucleic acid regions can be detected simultaneously with ease and sensitivity.

That is, the present invention includes a step of simultaneously amplifying a plurality of regions to be detected of different sizes in +11 sample DNA or RNA by the polymerase chain reaction method, (2) subjecting the amplified sample to electrophoresis, A method for detecting a nucleic acid region is provided, which includes the steps of: separating the plurality of amplified regions to be detected based on their respective sizes; and (3) detecting the separated nucleic acid fragments.

[Effects of the Invention] According to the method of the present invention, multiple nucleic acid regions can be detected simultaneously with high sensitivity. Therefore, in investigating genetic diseases and infectious diseases, checking the quality of vaccines and biological products, etc.
When it is necessary to detect multiple nucleic acid regions, this method is advantageous because it can be detected much more easily than conventional methods.

[Detailed Description of the Invention] In the first step of the method of the present invention, multiple nucleic acid regions to be detected are amplified by a gene amplification method. The gene amplification method itself is known as described in the above-mentioned literature. That is, a double-stranded nucleic acid is denatured to become a single strand, and two types of primers per region are hybridized to each strand, each hybridizing with the 5° end region of each strand of the region to be amplified. Using RNA polymerase, a complementary strand is synthesized using the above-mentioned brimer as a starting point to form a double strand, which is further denatured and hybridized with the above-mentioned brimer, and a complementary strand is synthesized using DNA or RNA polymerase. Repeat. If the cycle is repeated n times, the nucleic acid region is theoretically amplified 2'' times. The number of cycles is not particularly limited, but is usually about 20 to 40 times.

In the first step of the present invention, the characteristics are that a plurality of regions to be detected are simultaneously amplified and that the sizes of the plurality of regions to be amplified are made to be different. Simultaneous amplification of a plurality of regions can be performed by reacting the two types of primers described above for each region. That is, when detecting four regions, for example, it is sufficient to react with brimers that hybridize with the 5° terminal region of each chain in each region, that is, a total of '8 types of brimers. Furthermore, making the sizes of the plurality of amplified regions to be detected different can be achieved by selecting the base sequence of the primer. That is, the region to be amplified by the gene amplification method is sandwiched between a first brimer that hybridizes with the first strand of a double-stranded nucleic acid and a second brimer that hybridizes with the second strand of a double-stranded nucleic acid. Therefore, by selecting the base sequences of the first and/or second primer, the size of the region sandwiched between them can be arbitrarily changed.

In the method of the present invention, the sizes of multiple regions to be amplified are made different by selecting the base sequence of the primer. In this way, by making the amplification regions different in size, it becomes possible to easily separate the amplified nucleic acid molecules according to their sizes in the next electrophoresis step.

Next, the sample is subjected to electrophoresis to separate each amplified nucleic acid region. Separation of nucleic acid molecules by electrophoresis is well known in this field, and methods used in the Southern blot method and the Northam blot method can be applied as they are. As the gel, agarose or polyacrylamide gel is usually used. In this electrophoresis step, the plurality of regions to be detected that were amplified in the first step are separated based on their sizes.

Next, the separated nucleic acid fragments are detected. For example,
This can be carried out by transferring and immobilizing one separated nucleic acid molecule onto a substrate while maintaining the electrophoretic separation pattern, and detecting the transferred and immobilized nucleic acid fragments. These steps themselves are well known in this field, and the methods used in the Southern blot method and the Northan blot method can be applied as they are. That is, a nylon filter or a nitrocellulose filter can be preferably used as the substrate used here. Nucleic acid molecules can be immobilized, for example, by heat drying the filter or irradiating it with ultraviolet rays, using conventional methods. For example, detection of transcribed and immobilized nucleic acid molecules involves applying labeled probes that specifically hybridize to each amplified region, and determining whether these labeled probes specifically bind to the nucleic acid molecules on the substrate. By examining it, it can be known whether the nucleic acid region to be detected is present in the sample. The areas to be detected are fixed on the substrate while maintaining separate patterns according to their size, so it is easy to see at a glance which areas are present in which sample and which areas are missing. It's very convenient to know. Alternatively, after gel electrophoresis,
The gel is heat-dried without transferring and immobilizing the separated nucleic acid fragments onto a substrate, and the nucleic acid fragments can be detected while maintaining the separation pattern using a labeled probe in the same manner as described above.

The method of the present invention is a very basic technique and has an extremely wide range of application. can be applied to the diagnosis of

Furthermore, it can be applied to checking the quality of vaccines and other biological products by genetic analysis.

That is, for example, the surface structure of viruses such as the AIDS virus (HIV) often mutates, so there is a risk that vaccines once produced may become ineffective, but the method of the present invention allows rapid tracking of mutations in these viruses. becomes possible. Also.

When genetically engineering useful substances to be produced in microorganisms such as E. coli or in animal or plant cells, there is a risk that the region encoding the useful substance may mutate or drop out as the host cells proliferate. The method can be applied to rapidly identify such mutations and omissions in multiple regions. Furthermore, multiple nucleic acid molecules in a nucleic acid mixture containing multiple nucleic acid molecules can also be detected simultaneously. Therefore, the method of the present invention can be applied not only to detecting multiple regions in one type of nucleic acid molecule, but also to detecting regions in multiple different types of nucleic acid molecules.

Hereinafter, an example will be described in which the provirus of adult T-cell leukemia virus (hereinafter referred to as ATLV) was detected by applying the method of the present invention. Therefore, it can be applied in any case where it is desired to detect multiple nucleic acid regions, and its application is not limited to the detection of ATLV shown in the following example or the diagnosis of human diseases. That is clear.

[Example] Preparation of fil samples Lymphocytes were separated from 5 to 10 ml of peripheral blood from healthy individuals and ATL patients using a specific gravity solution, and DNA in the lymphocytes was extracted by a conventional method.

(2) Brimer and amplification region ATLV gag region (approximately 800th to 2090th base), pot region (approximately 2500th to 5180th base), env region (approximately 5180th to 6640th base), and pX region (approximately 6680th to 8360th base) The first region that overlaps with each of these regions for detecting
Chemically synthesized brimers having the nucleotide sequences shown in the table below, which specifically hybridize with the 5' terminal region of the strand and the second strand, respectively.In the table, the numbers on both sides of the nucleotide sequence of each brimer indicate the Hybridizing AT
The base positions of the LV genome are shown.

Therefore, for example, the 1375th to 1632nd bases are defined as the region for detecting the gag region, and this region is amplified. The size of each region amplified is gag
Area is 158 bp, pro/pol area is 20 b
p, env region is 193 bp, pX-IV is 220 bp
bp, and the size of each amplified region was different.

(3) Preparation of gene amplification method fil sample Take a part of the DNA sample prepared in (1), and
Final concentration in DTA(TE) buffer, lug/μ11.
: Melted. 50 μl of the mixture was heated at 95° C. for 5 minutes to denature DNA or RNA degrading enzymes that were expected to be present. After stirring at -degrees, the mixture was centrifuged at 6000 rpm for 30 seconds and used in the following operations.

Preparation of fii1 Reaction Reagent Mixture Reaction mixtures were prepared in a volume equal to or less than one test tube according to the number of samples.

10 x Tris/c1/MgclzM solution
10.1) Oμm dNTP mixture (4NTPs)
8.00 u 1gag area braai 7
-1 (0゜5u g/μm1 0.25 u 1gag
Area ply v-2fO, 5ug#zl) 0.25L
L1pol area bliv-1 (0,5ug/u 11
0.25 u 1pol area blur 1'? −2(0,
5+ug#zll 0.25ulenv area Brimer 110.5u g/μm1 0.25 u 1env
Area Brimer 2 ((1,5ug#zll 0.25
LL1px Area Brimer 1 (0.5ug/al
l 0.25 ulpx Area Brimer 2+0.
5μg/μl) 0.25μITaq polymerase [
51/u 11 0.50 u 1 total
20.50ti1 fiiiil gene amplified distilled water? 4.50 ul of the above reaction reagent mixture 20
．． 50 tLl and 5.00ul of the above DNA sample were mixed and centrifuged at 600 rpm+ for 30 seconds to precipitate pellets. After adding 2 drops of mineral oil, air bubbles were removed by centrifugation at 6000 rpm for 30 seconds. The centrifuge tube was set in a commercially available automatic temperature controller, and gene amplification was performed.

The temperature cycle was 1) 94°C for 2 minutes, 2) 60°C,
3) 72°C for 13 minutes. After completion, the samples were stored at 4°C.

(4) Electrophoresis 1% Ultra Pure Agarose (manufactured by BRL), 2% N
Usive GTG agarose (manufactured by FMC Bioproducts) and 0.3 μg and 7 ml of ethidium bromide were heated and dissolved in 1×TE buffer to prepare an agarose gel. 6x loading buffer was added to the amplified sample obtained in (3), and 10μl of it was placed in each well of the gel, and electrophoresis was performed (50mA, 2-
3 hours), and the buffer containing the marker was also subjected to electrophoresis under the same conditions.

(5) DNA transcription by Southern blotting According to a conventional method, the DNA electrophoretically separated in (4) was transferred to a nylon filter.

(6) 5' end labeling of oligo DNA probes Oligo DNA probes for detecting each region that have the base sequences shown in the table above (in the table, the numbers on the left and right of the base sequence of the probe indicate that the probe is hybridized). The 5' end of the ATLV genome (location in the ATLV genome shown) was labeled with 32p. Labeling was performed by incubating a reaction mixture with the following composition at 37° C. for 1 hour.

Mixed oligo DNA probe (0.5μg/μm10.6
μl cold distilled water 5.4μl
r −”P −ATP (−3000Ci/mmol
l 10. Ou 1 total 20. Ou
1 After the reaction, it was separated using a Quick 5pin column (G25 Sephadex column) (manufactured by Boehringer-Mannheim) (Centrifugation 3 (100 rpm, 2 minutes) (71 DNA band "detection filter") Prehybridization 5 x 5SPE, 5X Denhartz solution and 0.5%S
10 x 20 cm2 with 20+ml drops of OS
Filters were treated and incubated for 2 hours at 56°C.

(Iil hybridization fit's liquid after Brepipe hybridization and ff2P
- Probe (2x 10' dpm/1.4-5 m
l/10 x 20 cm2 filter) and 5
Incubate for 2 hours at 6°C.

fiiiil cleaning The following cleaning was performed.

112 x 5SPE10.1%SOS, room temperature, 10
Minutes 2) Same as above 315 x 5SPE10.1%SOS at 55℃, 10
Autoradiography was carried out for 1 to 2 days using a conventional minute-fivl development method.

(8) The results are shown in the figure. In the figure, lane 1 is the DNA of healthy subjects, and lanes 2 to 11 are peripheral blood lymphocyte DNA from ATL patients.
The results for A are shown. Lane 12 is a marker ($
1lae III digest of X 174, fragment size is 1
357 bp, 1078 bp, 372 bp, 603
bp, 310 bp, 281 bp, 271 bp, 2
34 bp, 194 bp, 118 bp and 72 bp
bp) is shown. As you can see from the diagram,
In lane 1, no expression of ATLV was observed;
The ATL patient in lane 2 expresses the entire ATLV region, the patient in lane 3 expresses only the gag region, the patient in lane 4 expresses only the pal region, the patient in lane 5 expresses only the env region, and the patient in lane 6 patients in lane 7 express only the gag and pal regions; patients in lane 8 express only the gag and env regions; patients in lane 9 express only the gag and pX regions; It can be seen that the IO patient expresses only the pol region and env region, and the patient in lane 11 expresses the gag region, pal region, and env region.

As is clear from the above examples, according to the method of the present invention, it is possible to simultaneously and easily detect multiple nucleic acid regions to be detected with high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS The figure shows autoradiography obtained as a result of detecting ATLV provirus by applying the method of the present invention.

Claims (1)

[Claims] A step of simultaneously amplifying a plurality of regions to be detected with different sizes in a sample DNA or RNA by a gene amplification method, and subjecting the amplified sample to electrophoresis to perform the amplified detection region. A method for detecting a nucleic acid region, the method comprising: separating a plurality of regions based on their respective sizes; and detecting the separated nucleic acid fragments.