JPS63241465A - Dna probe - Google Patents

Abstract

PURPOSE:To shorten the time required for hybrid and to improve hybrid effi ciency and labeling efficiency by forming a titled probe in such a manner that the heterologous single chain DNA fragments complementary with the different sections in the DNA or RNA to be detected are respectively labeled. CONSTITUTION:The heterologous single chain DNA fragments complementary with the different sections in the DNA or RNA to be detected consist respectively of the labeled plural heterologous DNA fragments. This DNA or RNA is the DNA or RNA derived from microorganisms such as hepatitis virus, AIDS virus, herpes virus and animal and vegetable cells in which the individual fragments have complementarity overall with the base sequence of DNA, RNA in a widest possible range. The resulted single chain can be labeled by an ordinary method. The DNA or RNA of the virus, etc., is detected by using this probe. Then, the time required for the hybrid is shortened and the hybrid efficiency and the labeling efficiency are improved.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a reagent, ie, a DNA probe, for detecting or quantifying DNA or RNA of viruses, microorganisms, or animal and plant cells.

<Prior Art> Originally, the base sequence of DNA or RNA is unique to each virus species and biological species. Then, that DNA or RNA specifically forms a double strand, that is, hybridizes, with the complementary NA or RNA. Taking advantage of this property, DNA probes have recently come to be used as reagents for detecting or quantifying DNA or RNA.

Conventionally, DNA probes are prepared by labeling DNA complementary to DNA or RNA derived from a virus, microorganism, or animal or plant cell to be detected with a labeling agent. The DNA used includes viruses, microorganisms,
D that has been cloned by incorporating DNA itself derived from animals or plants, or all or part of it into a vector.
NA and DNA are synthesized from scratch. In many cases, 1
It is a double-stranded DNA with a length of 000 base pairs or more, and the synthetic DNA is a single-stranded DNA with a length of 100 base pairs or less.

On the other hand, radioactive isotopes are generally used as labels, but recently M elements have been identified using hyotine-ahidine bonds. In other words, the molecular weight of egg white, etc. is 68,
Since ahidin, a basic protein with a molecular weight of 000, and biotin with a molecular weight of 244 do not exhibit a strong affinity with an affinity constant of -+o15M-1, they are complementary to the DNA or RNA to be detected, and NA is unlikely to inhibit the complementarity. DNA or RNA to be detected by labeling with low molecular weight biotin
Enzyme labeling is performed by hybridizing the enzyme-avidin complex with biotin-avidin. Furthermore, labeling using antigen-antibody reactions has also been carried out. That is, biotin, fluorescein,
DNA or RNA to be detected by labeling an antibody against DNA bound with a hapten such as N-acetoxy-2-acetylaminofluorene with fluorescence or an enzyme.
This is a method of detecting the target DNA or RNA by hybridizing the hapten-bound DNA with the DNA or RNA to be detected, and then binding the labeled antibody through an antigen-antibody reaction.

<Problems to be Solved by the Invention> Conventional DNA probes are single-stranded DNA or double-stranded DNA bound to synthetic DNA, labeled double DNA, or ff1 DNA; Usually 100
It is DNA with 0 base pairs or more. When using a double-stranded probe, it is necessary to denature it with alkali or heat to make it a single strand before hybridizing it with the DNA or RNA to be detected. Furthermore, since this is necessary for long DNAs of 1000 base pairs or more, it takes a long time for hybridization to occur. Furthermore, hybridization between probes occurs due to the coexistence of mutually complementary strands, reducing the efficiency of hybridization with DNA or RNA to be detected.

On the other hand, synthetic NA does not have the above disadvantages, but
Labeling efficiency is extremely low because the DNA strand is short.

The present invention eliminates the drawbacks of conventional DNA probes, and provides short time required for hybridization, high hybrid efficiency, high labeling efficiency, and simple operation.
The purpose is to provide A probe.

Means to solve problems〉 The above object is achieved by the present invention as described below.

That is, the present invention can detect DNA or RN.
A heterologous single 1' that is complementary to a different site in A! The present invention provides a DNA probe consisting of a plurality of labeled heterologous DNA fragments, each of which is labeled.

The DNA or RNA to be detected by the present invention is not particularly limited, but specifically, hepatitis (type A, type B) virus, AIDS virus (HIV-nl) AT Lwi/L
, HIV-I > , H/L, pes (herpes simplex, varicella, seastar) virus, cytomegalovirus, measles virus, rubella virus, polyoma virus, papilloma virus, SV40 virus, coxalaki virus, Echovirus, influenza virus, rabies virus, yellow fever virus, Japanese encephalitis virus, Marburg virus, adenovirus, dengue virus, EB virus, mambus virus, cotton sinia virus, parvovirus, coxalaki virus, rotavirus, tanapox Viruses, Yaba virus, Lassa fever virus, tobacco mosaic virus, mycoplasma, Tsutsugamushirikedcha, Q-heating Kecha, rash, Chlamyzoia trachomanis, Chlamydia apsittacosis, Phosphorus bacteria, Clostridium tetani, Staphylococcus aureus, Streptococcus , Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus anthracis, Streptococcus pneumoniae, Salmonella enterica, Vibrio cholerae, Salmonella typhi,
Salmonella paratyphi, Clostridium botulinum, Purcella, Shigella, Vibrio parahaemolyticus, Yersinia pestis, Escherichia coli, Campylobacter,
DNA or RNA derived from viruses such as Candida, toxoplasma, malaria, syphilis, lung cancer cells, cancer cells, microorganisms such as bacteria, fungi, and protozoa, and animal and plant cells.
A, and it does not matter whether it has the entire base sequence, a fragment, a double strand, or a single strand.

The DNA probe according to the present invention is characterized by high hybridization efficiency and high labeling efficiency, and is characterized by high hybridization efficiency and high labeling efficiency.
It is desirable that the individual fragments comprehensively have as wide a range of complementarity as possible to the base sequence of A or RNA, and most preferably the DNA or RNA that is to be detected.
It must be continuously complementary to the entire [J sequence of A, but it may be one region or multiple regions of at least 1000 salt bases, within which each fragment is continuously complementary. It does not matter if they are intermittent or complementary. Therefore, the number of fragments is preferably as large as possible, and although it depends on the length of the probe, it is 3 or more, more preferably 5 or more. Furthermore, the size of the -fragment is preferably about 100 to 500 bases, more preferably about 150 to 300 bases.

The method for preparing the single-stranded DNA of the present invention is not particularly limited, but includes, for example, a method of cleaving single-stranded DNA cloned with a batatteriophage M13 vector using ultrasound, or using the single-stranded DNA as a template and using multiple primers. A method in which the DNA is elongated with an enzyme and then separated from the template DNA (patent application 1986).
2-30/120), etc.

The single-stranded DNA thus obtained can be labeled using a conventional method. That is, it may be directly recognized by a labeling agent such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, or an enzyme, or it may be recognized by biotin, N-acetoxy-2
- A low-molecular substance such as acetylaminofluorene may be bound and labeled with an antibody using such a low-molecular substance as a hapten, or a complex of a substance such as ahidin and a labeling agent. Specifically, for example, single-stranded DNA is treated with Futobiodin (Biotechnology Research Enterprises) or Chemiprobe (Orgenics), or 3-stranded DNA is used as a substrate during DNA elongation.
Labeling can be easily carried out by methods such as using a radioisotope such as 32P or 125■, or a base bound to a hapten or the like.

The DNA or RNA of viruses, microorganisms, animal and plant cells can be detected using the DNA probe of the present invention, for example, by the following method. First, a sample such as tissue or body fluid in which viruses or microorganisms are thought to exist, animal or plant cells, cancer cells, etc. is fixed on a glass plate.

Or DNA or R extracted from tissues, body fluids, or cells.
NA is immobilized on a filtration membrane such as nitrocellulose or nylon. Next, the DNA or RNA to be detected is denatured to become a single strand. By incubating this with the DNA probe of the present invention, the D.
Hybridize the DNA probe with NA or RNA. If no labeling agent has been bound in advance, the hybridized DNA probe is labeled with an enzyme or the like, and detected by color development of the labeled enzyme with a substrate. Note that each step usually includes a cleaning step.

In addition, a method using electrophoresis (patent application No. 61-9787)
7> can also be detected. In this method, the DNA probe of the present invention is added to a solution of denatured DNA or RNA to be detected, hybridized, and then a
Electrophoresis is performed perpendicularly to the surface of a gel layer of ~3# agarose, polyacrylamide, etc., and the DNA or RNA to be detected that has hybridized with the DNA probe is isolated with 1ff.
i-t must be kept inside or not hybridized D
The NA probe is a method of detecting DNA probes remaining on or inside the gel layer by passing through the gel layer and removing it.

Example of implementation EXAMPLES Hereinafter, the present invention will be roughly explained in detail with reference to Examples.

Example 1 (1) M incorporating hepatitis B virus (HBv) DNA
"Preparation of 13rnp19 single-stranded DNA (IB/M13)" Cloning with M13 phage and Dide.

xy sequence method” (Amashi Vmu Japan Co., Ltd. 1)
HBV restriction enzyme,
HB/M with 1.4 Kb fragment incorporated by BamHI
I got 13.

(2) Cleavage of IB/M13 Cloning of IB/M13 DNA in the M13rnp19 DNA base sequence Restriction enzyme of I night Find ■ Base sequence containing recognition site AAGCTTGCATGCCTG
Complementary NA oligo? -(50.[) at 260 μm,
/ml> 15μg and Ha/M13 (0°5μ9/
LMgCfJ2, which was mixed with >100 μg and incubated at 55°C for 5 minutes and then at subtemperature for 5 minutes, was
m) l, add NaCF to 60mM, t-1
ind I [I (10 units /ufJ) 8
μg was added and incubated at 37° C. for 3 hours. Cleavage was confirmed by electrophoresis.

(3) t-1indl of ToIBV Yamaki DNA in elongated Ha/M13
A 15-base DNA oligomer GCGGCT complementary to the base sequence at an interval of about 200 bases from the cut end by II.
AGGAGTTCF, CCTGTTTAGCTTGTA
, ATGTAGCCCATGAAG, AATGGCAC
TAGTAAA, TGG△ATTAGAGGACA, T
TGAGAG△△GTCCAC (each 50.
0. /d) was mixed with the solution of cleaved ring-opened HB/M13 obtained in (2) and incubated at 55°C for 5 minutes and at room temperature for 5 minutes.
Buffer solution containing P, dCTP, dGTP, dTTP (67
...■ Potassium phosphate, 6.7mM9CN2, DI+
=7. /l) 100 μg and 5-aminoaryl-dUTP (P.

R,tanger et al., Proc. Natl.
8cad, Sei, USA, 78°663
3 (1981)] was added to 10 μg of a 1.8 mM aqueous solution of DNA Polymerase Large Fragment (4,2 Unit S/VIG) 11, and incubated at 25° C. for 5 minutes. 250m)!
Add 50 μg of EDTA aqueous solution (pH = 7.0)
1olccular Cloning (Cold S
pring Harbor Laboratory 1
DNA was recovered by phenol extraction and ethanol precipitation according to 982).

(4) Separation and recovery of elongated DNA A 3% agarose gel membrane (17 mm x 2 mm) was provided with small chambers partitioned by semipermeable membranes on both sides, and the DNA solution obtained in (3) was placed on one side (50 m). INAO
Hllmt (EDTA) on the other hand and electrophoresis solution (30
mH1N a OHll mHE D T-A > was filled, electrophoresis was performed in the electrophoresis solution (70 V for 20 minutes) with the DNA solution side as the cathode, and an elongated DNA solution was obtained from the small chamber on the anode side. 200 by marker by electrophoresis
It was confirmed that the base of the DNA was 1'''.

(5) Biotin labeling The obtained DNA solution was dialyzed in a 0.1H NaHCO3 aqueous solution. ) Layer line and set the total set to 900μρ, Biot
in-X-NH3 (Behring Diagnost
ics La Jotla, CA92037) 10t
25 μg of l'j/ufJ DMF was added and stirred at room temperature for 1 hour. The reaction solution was diluted with TE buffer (10ml) Tris.
-tllll, 1mMED l-A, l)l+
= 8.

The biotin-labeled DNA was removed by dialysis in O.

<Effects of the Invention> The DNA probe according to the present invention is an excellent probe in that the time required for hybridization is short, the hybridization efficiency is high, and the labeling efficiency is high. Furthermore, since it is a single chain, it does not need to be denatured and has the advantage of being easy to use.

Claims (1)

[Claims] (1) A DNA probe consisting of a plurality of labeled heterologous DNA fragments, in which each heterologous single-stranded DNA fragment complementary to a different site in the DNA or RNA to be detected is labeled.