GB2239948A - Nucleic Acid Assay - Google Patents
Nucleic Acid Assay Download PDFInfo
- Publication number
- GB2239948A GB2239948A GB9027423A GB9027423A GB2239948A GB 2239948 A GB2239948 A GB 2239948A GB 9027423 A GB9027423 A GB 9027423A GB 9027423 A GB9027423 A GB 9027423A GB 2239948 A GB2239948 A GB 2239948A
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- United Kingdom
- Prior art keywords
- naphthol
- och3
- derivative
- phosphate
- phosphatase
- Prior art date
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- 238000007826 nucleic acid assay Methods 0.000 title 1
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 33
- 239000010452 phosphate Substances 0.000 claims abstract description 33
- -1 naphthol AS derivative phosphate Chemical class 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 20
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 20
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 20
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims abstract description 10
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims abstract description 10
- 230000001678 irradiating effect Effects 0.000 claims abstract description 5
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 4
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 9
- 238000001514 detection method Methods 0.000 description 18
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 11
- 239000002244 precipitate Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 239000000523 sample Substances 0.000 description 6
- JFGQHAHJWJBOPD-UHFFFAOYSA-N 3-hydroxy-n-phenylnaphthalene-2-carboxamide Chemical class OC1=CC2=CC=CC=C2C=C1C(=O)NC1=CC=CC=C1 JFGQHAHJWJBOPD-UHFFFAOYSA-N 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 150000001448 anilines Chemical class 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- ALKYHXVLJMQRLQ-UHFFFAOYSA-N 3-Hydroxy-2-naphthoate Chemical compound C1=CC=C2C=C(O)C(C(=O)O)=CC2=C1 ALKYHXVLJMQRLQ-UHFFFAOYSA-N 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 2
- MKARNSWMMBGSHX-UHFFFAOYSA-N 3,5-dimethylaniline Chemical compound CC1=CC(C)=CC(N)=C1 MKARNSWMMBGSHX-UHFFFAOYSA-N 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 150000003931 anilides Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- FAIAAWCVCHQXDN-UHFFFAOYSA-N phosphorus trichloride Chemical compound ClP(Cl)Cl FAIAAWCVCHQXDN-UHFFFAOYSA-N 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 241000252203 Clupea harengus Species 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000001769 aryl amino group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019514 herring Nutrition 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- CNXZLZNEIYFZGU-UHFFFAOYSA-N n-(4-amino-2,5-diethoxyphenyl)benzamide Chemical compound C1=C(N)C(OCC)=CC(NC(=O)C=2C=CC=CC=2)=C1OCC CNXZLZNEIYFZGU-UHFFFAOYSA-N 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/094—Esters of phosphoric acids with arylalkanols
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method for assaying nucleic acids or a sequence thereof comprises binding a sample such as a nucleic acid to phosphatase; reacting the phosphatase with a naphthol AS derivative phosphate; and irradiating the reaction product with excited light and detecting a fluorescence emitted therefrom.
Description
I- ::2!:3 'D ED -el C51 1 - METHOD FOR ASSAYING NUCLEIC ACIDS USING
NAPHTHOL AS DERIVATIVE PHOSPHATE
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
1 The present invention relates to a method for assaying nucleic acids which can efficiently detect nucleic acids, etc. by fluorescence. RELATED ART In medical and biological fields, a means for labeling, for example, a DNA (or RNA) probe with a radioactive isotope, hybridizing the labeled Probe with a target nucleic acid and then detecting the target nucleic acid by autoradiography has been currently performed widely.
However, the isotope method involves many drawbacs which are serious obstacles to application and development of this technology, The drawbacks of the isotope method are as follows.
(a) In nucleic acid hybridization, the method lacks any spatial resolution sufficient to reveal relative positional relationship between contiguous signal.
(b) Experimental procedures using isotopescan only 2 be carried out in isotope laboratories equipped with special facilities. This is a cause for hindering application of the hybridization method espdcially to clinical inspection.
(c) Use of isotopesis dangerous for laboratory workers-even in laboratories. in addition, a danger for ordinary people also always exists because of wastes, etc.
(d) A long time (several weeks to several months) may be required for detection, so application to rapid clinical diagnostication is difficult.
(e) Radioactivity decays with a definite half-life period so that experiments should be scheduled to fit a purchase date of isotope. If the schedule chart is slightly altered, there would be a danger of wasting isotope or experimental results in a large scale.
(f) In order to enhance detection sensitivity, it is required to incorporate radioactivity to a nucleic acid probe as high as possible. However, the nucleic acid labeled enough to increase its radioactivity easily suffers from radioactive disintegration.
(g) In general, isotope is extremely expensive and it is not unusual to use isotope worth several hundred yen in one run. This prevents general spread of the hybridization method.
In view of such background, some DNA or RNA labeling methods in place of radioactive isotope have been developed so far. For example, BLU GENE KIT commercially available from Besesda Research Laboratories
Inc. (BRL Inc.) is known. Furthermore, "Nucleic acid probe and use thereof" is disclosed in Japanese Patent Application Laid-Open No. 60226888.
However, these techniques merely eliminate a part of the drawbacks described above. In particular, detection sensitivity is not comparable to that of the isotope method.
That is, in the above labeling, the detection sensitivity is,10-12 g DNA" and slightly inferior to "10-13 g DNA" in the isotope method.
In view of such problems, an object of the present invention is to provide a method for assaying nucleic acids which solves the drawbacks of safety precautions, etc. in the isotope method and provides excellent detection sensitivity.
SUMMARY OF THE INVENTION
The present invention provides a method for assaying nucleic acids or the like which comprises binding phosphatase to a sample such as nucleic acids, etc., reacting the phosphatase with a naphthol AS derivative phosphate, then irradiating the reaction products with an excited light and detecting fluorescence emitting therefrom.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
It is most notable in the present invention to use a naphthol AS derivative phosphate to which phosphatase is bound and to detect fluorescence emitted by an excited light.
1 Examples of the phosphatase include alkali phosphatase, acid phosphatase, etc.
The detection of a sample in the present invention includes detection of nucleic acid (DNA or RNA), detection of protein, immunological detection of a chemical compound using antibody, etc.
An example of the naphthol AS derivative phosphate includes the basic skeleton shown by Formula [I].
Formula [ 11 0 11 0 P (0 H) 2 CnCONH-X In Formula [I] described above, X represents on of the following formulae.
CH3 -0, CH3 CH3 -. CH3 OCH3 OCH3 a i j - 5 OCH3 -c OCH,3 OCH3 -0 OCH3 OCH3 N COCH3 -co CH,3 - - -CH3 CH3 3 -GCH=CHOCH's 6 For preparing the naphthol AS derivative phosphate, there are, for example, a process explained below.
The process for preparing a naphthol AS derivative phosphate, which comprises boiling 2-hydroxy-3-naphthoic acid shown by:
OH L,CWH and arylamino derivative with phosphorus trichloride in the presence of toluene or xylene to give a naphthol AS derivative shown by the formula:
Formula II 01 OH CCONH-X (wherein X represents the same as in the Formula [I]), and then reacting the naphthol AS derivative with phosphorus oxychloride in pyridine.
In the method for assay in accordance with the present invention, the naphthol AS derivative phosphate is reacted with the phosphatase described above followed by irradiating with an excited light, whereby the dephosphating product of the naphthol AS derivative phosphate emits fluorescence. Then, the emitted fluorescence can be detected.
i 1 1 j The aforesaid naphthol AS derivative phosphate is reacted with the phosphatase combined with a sample (e.g. nucleic acids) on a membrane filter made of nylon so as to produce a dephosphating product of the naphthol AS derivative phosphate, which adheres to the nylon membrane filter and displays fluorescence. Then, the fluorescence and the pattern thereof (spots, and bands produced by electrophoresis) are detected by irradiating with the excited light.
In the present invention, intense fluorescence can be obtained by the use of the naphthol AS derivative phosphate described above so that detection sensitivity can be improved; for example, 10- 13 g of DNA is detectable.
In the present invention, no isotope is used and therefore, the drawbacks of the prior art can be removed.
Thus, according to the present invention, a method for assaying nucleic acids, etc. which can provide excellent detection sensitivity can be presented.
Further, according to the method of the present invention, the dephosphating product of the naphthol AS derivative phosphate can be produced in high yield. DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1
For the purpose of verifying the effectuality of the naphthol AS derivative phosphate as a probe for nucleic acids, DNA Labeling and Detection kit of Boehringer Mannheim, and naphthol AS-3,5-dimethylphenyl phosphate of Example 2 described later were used to detect DNA on a nvlon membrane filter.
DNA was labeled with digoxigenin (Dig), diluted and spotted on the nylon membrane filter. Each.3f the spots included DNA of herring spermatozoa in the amount of 50 ng (50 x 10- 9 g) as DNA of no peculiarity.
The aforesaid experiment was conducted on 0.08 to 25 pg of Dig-labeled DNA as shown in Fig. 1. 0 pg in the figure shows a blank test. The test results are shown in Fig. 1. Reference numeral 1 designates a carrier filter for a specimen of nucleic acids, and 11 designates fluorescence sensitized portions. "+" represents a fact that the DNA can be detected. " " represents that the DNA cannot distinctly be detected. "-" represents that DNA cannot be detected. As apparent from the above, DNA could satisfactorily be detected even in the small amount of 0.4 pg. In the amount of 0.08 pg, satisfactory detection was impossible.
Then, using the smaller amount of the aforesaid DNA, another experiment was conducted on 0.0156 to 0.5 pg of Dig-labeled DNA in the same manner as described above. The test results are shown in Fig. 2 similarly in Fig. 1. As shown in Fig. 2, satisfactory detection could be attained in the 0. 125 pg (125 fg). In the amount of 0.0625 pg, detection was not satistactory.
In this former experiment, a conventional color development detection using azo-color, Fast Blue BB (of POLYSCIENCE, INC.) was conducted. As the result of this detection, the detectable spot included 0.5 pg (0.5 x 10- 12 g) of the DNA. Example 2 A naphthol AS derivative phosphate of the present invention was produced by the following proceigses.
According to the description of Enzyme Histochemistry, 5 gr (0.027 mol) of 2-hydroxy-3-naphthoic acid, 40 ml of dehydrated xylene and 0.023 mol of 3,5dimethyl aniline were stirred in a 100 ml NASU flusk provided with Graham condenser at 80 'C for 10 minutes.
Then, 0.01 mol of phosphorous trichloride was added thereto. The resulting mixture was refluxed for 2 hours. Thereafter, the reaction solution was decanted as in hot state to skim the supernatant fluid. After cooling the fluid at 4 'C, it was subjected to filtration. The precipitates thus obtained was eluted with xylene and then water. Further, the precipitates was neutralized with 2% aqueous solution of sodium carbonate, and xylene was removed therefrom by boiling.
Then, the precipitates was rendered pH = 9 with 2% aqueous solution of sodium carbonate, filtrated and cooled. The precipitates thus obtained was eluted with water. The precipitates was added to 3% HC1 solution, heated, filtrated and then cooled. Then the precipitates was washed with hot water and dried.
- Next, the precipitates were recrystalized to produce 3-hydroxy-2naphthoic acid V,V-dimethyl anilide shown by the following formula.
ta C'ONH H3 X CH3 1 g of this naphthol AS derivative was dissolved in 8 ml of pylidine. After stirring this solution at 0 C for 30 minutes, phosphorus oxychloride (2.5 eq) cooled similarly was added thereto and stirred at 0 'C for 4 hours. Then, ice was added to the solution to terminate the reaction.
The reaction product thus obtained was purified on a reverse phase silica gel column and then a normal phase silica gel column to produce 3-hydroxy2-naphthoi acid- V,5'-dimethyl anilide phosphate shown by the following formula.
0 11 OP (OW 2 CH3 CONH-0 CH3 Examples 3 through 12 Various naphthol AS derivative phosphate were produced using various aniline derivative in the same manner as Example 2.
C - 11 Detection test similar to Example 1 was conducted on each of the naphthol AS derivative phosphate by the same method as in Example 1, i.e. spolting on the carrier filter for a specimen of nucleic acids.
The aforesaid aniline derivative and naphthol AS derivative phosphate obtained therefrom are shown in Table 1.
The test results are shown in Table 2. As apparent from Table 2, the extremely small amount of DNA can be detected in high sensitivity..
The naphthol AS derivative phosphate shown in Examples 2 through 12 are as follows.
Example 2: 3-hydroxy-2-naphthoic acid-3',5'dimethyl anilide phosphate Example 3: 3-hydroxy-2-naphthoic acid-2',4'dimethyl anilide phosphate Example 4: 3-hydroxy-2-naphthoic acid-V-chloro4',6'dimethoxyanilide phosphate Example 5: 3-hydroxy-2-naphthoic acid-3',V,5'trimethoxyanilide phosphate Example 6: 3-hydroxy-2-naphthoic acid-V-aminoanthracene phosphate Example 7: 3-hydroxy-2-naphthoic acid-3't5'-dimethoxyanilide phosphate.
Example 8: 3-hydroxy-2-naphthoic acid-2',4',6'trimethylanilide phosphate i i i Example 9: 3-hydroxy-2-naphthoic acid-V-acethylamino anilide phosphate Example 10: 3-hydroxy-2-naphthoic acid-V-aminonaphthalene phosphate Example 11: 3-hydroxy-2-naphthoic acid-2,5'-dimethoxy-4'-stylbeneamino phosphate Example 12: 3-hydroxy-2-naphthoic acid-4'-cyanoanilide phosphate 1 Table 1 (1)
EXAMPLE aniline derivative naphthol AS derivative 0 11 H2N CH3 CH3 CM3 0 OCH3 11 0 oci- PaN- OCH3 CONH OCH3 c C2 1 W 1 Table 1.(2)
EXAMPLE
6 aniline derivative naphthol AS derivative 0 O'C H 3 11 OPCOHh OCH3 "-Q0CHS 1 CONH - OCH3 OCH3 - OC"3 0 a H2N Z, P(OH)2 WA;-, CONH--= 1 1 # 1 Table 1 (3)
EXAM 7 8 1 aniline derivative OCH3 H2N-O OCH3 CH3 H2N-01-CH3 CH3 naphthol AS derivative 0 a KO 2 OCH3 CONH OCR3 0 1 O'(OH)2 CONH - YCH3 CH3 ul t Tab;1e 1 (4) EXAMPLE aniline derivative NHCOCH3 9 H2N-C H2N-CO 1, 'A, naphthol AS derivative 0 Wz TCOH 5>, b NHCOCH3 Z:-" 'It CONH-.
0 11 M1 'I OKOW2 "!:
CONH-W 1 Table 1 (5)
EXAMPLE
11 aniline derivative QW3 H2N-aCH=CH- CCHS 12 1 H2N W naphthol AS derivatve a OCH3 C"-F\---CH -2L 0 11 COH)2 CONH- W 1 Table 2 (1)
Dig-labeled 0 0.()6 0.125 0.25 0.5 1 2 10 25 NA (pg r:E:XAMPLEi= (pg) 3 + + + + + + + + 4 + + + + + + + + 6 7 + + + + 3 + + + + 1, 1 -A 00 1 4 Table 2 (2)
Dg-labeled 0.06 0.125 0.25 0.5 1 2 10 100 200 ig-labeled DNA (pg 0 rEXAMPLE DNA (p ' - 9 + + 12 --- 1 1 -A W 1
Claims (2)
- What is claimed is:A method for assaying nucleic acids or the like which comprises:binding a sample such as a nucleic acid to phosphatase; reacting the phosphatase with a naphthol AS derivative phosphate; and irradiating the reaction product with an excited light and detecting a fluorescence emitted therefrom.
- 2. A method for assaying nucleic acids or the like according to claim 1, wherein said naphthol AS derivative phosphate is represented by the following formula:0 11 1 OPOk CONH-X Formula [ 11 wherein X represents one of the following formulae, CH.3 CH3 CH.3 -1 OCH3 -: \ OCH3 CL OCH.3 OCH3 OCH,3 C H.3 OCH.3 CH,3 -}: -CH,3 CH3 1 -- 22 - WCOCR3 -C -co OCH3 -GCH=CH- 1 Published 1991 atIbe Patent 0111ce. State House. 66/71 High Holborn. London WCIR47P. Further copies may be obtained from Sales Branch. Unit 6. Nine Mile Point. Cwmfelinfach. Cross Keys. Newport. NPI 7HZ. Printed by Multiplex techniques ltd. St Mary Cmy, Kent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34315389 | 1989-12-28 | ||
| JP23222390A JPH03251200A (en) | 1989-12-28 | 1990-08-31 | Determining nucleic acid or the like with naphthol as phosphoric acid |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB9027423D0 GB9027423D0 (en) | 1991-02-06 |
| GB2239948A true GB2239948A (en) | 1991-07-17 |
| GB2239948B GB2239948B (en) | 1994-01-12 |
Family
ID=26530342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9027423A Expired - Fee Related GB2239948B (en) | 1989-12-28 | 1990-12-18 | Fluorescence assay using phosphatase and a naphthol phosphate derivative |
Country Status (2)
| Country | Link |
|---|---|
| DE (1) | DE4041880C2 (en) |
| GB (1) | GB2239948B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2246859A (en) * | 1990-08-07 | 1992-02-12 | Aisin Seiki | Method for assaying nucleic acids |
| GB2250991A (en) * | 1990-12-21 | 1992-06-24 | Aisin Seiki | Naphthalene derivatives |
| GB2270077A (en) * | 1992-08-28 | 1994-03-02 | Aisin Seiki | DNA sequencing method using a fluorescent substrate |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3844485A (en) * | 1984-02-09 | 1985-08-15 | Enzo Biochem Inc. | Heterologous detection of cabeled dna |
-
1990
- 1990-12-18 GB GB9027423A patent/GB2239948B/en not_active Expired - Fee Related
- 1990-12-27 DE DE19904041880 patent/DE4041880C2/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2246859A (en) * | 1990-08-07 | 1992-02-12 | Aisin Seiki | Method for assaying nucleic acids |
| GB2246859B (en) * | 1990-08-07 | 1994-08-17 | Aisin Seiki | Assaying of nucleic acids or proteins using a naphthol phosphate derivative |
| US5532125A (en) * | 1990-08-07 | 1996-07-02 | Aisin Seiki Kabushiki Kaisha | Method for assaying nucleic acids |
| GB2250991A (en) * | 1990-12-21 | 1992-06-24 | Aisin Seiki | Naphthalene derivatives |
| GB2250991B (en) * | 1990-12-21 | 1994-08-10 | Aisin Seiki | Naphthalene derivatives and their use in methods for assaying nucleic acids |
| GB2270077A (en) * | 1992-08-28 | 1994-03-02 | Aisin Seiki | DNA sequencing method using a fluorescent substrate |
| GB2270077B (en) * | 1992-08-28 | 1996-09-25 | Aisin Seiki | DNA sequencing method using a fluorescent substrate |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2239948B (en) | 1994-01-12 |
| GB9027423D0 (en) | 1991-02-06 |
| DE4041880A1 (en) | 1991-07-04 |
| DE4041880C2 (en) | 1994-03-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19971218 |