DE19950823A1 - Quantitatives Verfahren zum Analysieren einer Targetnukleinsäure - Google Patents
Quantitatives Verfahren zum Analysieren einer TargetnukleinsäureInfo
- Publication number
- DE19950823A1 DE19950823A1 DE1999150823 DE19950823A DE19950823A1 DE 19950823 A1 DE19950823 A1 DE 19950823A1 DE 1999150823 DE1999150823 DE 1999150823 DE 19950823 A DE19950823 A DE 19950823A DE 19950823 A1 DE19950823 A1 DE 19950823A1
- Authority
- DE
- Germany
- Prior art keywords
- primer
- nucleic acid
- pcr
- target nucleic
- molecules
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 62
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 60
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 60
- 230000003321 amplification Effects 0.000 title claims abstract description 35
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 35
- 239000012472 biological sample Substances 0.000 title claims abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 81
- 238000005259 measurement Methods 0.000 claims abstract description 49
- 239000012085 test solution Substances 0.000 claims abstract description 11
- 230000000295 complement effect Effects 0.000 claims abstract description 3
- 239000013615 primer Substances 0.000 claims description 155
- 239000002987 primer (paints) Substances 0.000 claims description 155
- 239000003550 marker Substances 0.000 claims description 39
- 238000001514 detection method Methods 0.000 claims description 32
- 239000002773 nucleotide Substances 0.000 claims description 16
- 125000003729 nucleotide group Chemical group 0.000 claims description 16
- 238000005311 autocorrelation function Methods 0.000 claims description 13
- 230000003287 optical effect Effects 0.000 claims description 11
- 239000007850 fluorescent dye Substances 0.000 claims description 8
- 238000009396 hybridization Methods 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 7
- 238000004445 quantitative analysis Methods 0.000 claims description 7
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 7
- 239000000975 dye Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000033001 locomotion Effects 0.000 claims description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims description 2
- OHOQEZWSNFNUSY-UHFFFAOYSA-N Cy3-bifunctional dye zwitterion Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C2=CC=C(S(O)(=O)=O)C=C2C(C)(C)C1=CC=CC(C(C1=CC(=CC=C11)S([O-])(=O)=O)(C)C)=[N+]1CCCCCC(=O)ON1C(=O)CCC1=O OHOQEZWSNFNUSY-UHFFFAOYSA-N 0.000 claims description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 2
- -1 Cy 3.5 Chemical compound 0.000 claims 1
- 239000000523 sample Substances 0.000 description 52
- 238000002060 fluorescence correlation spectroscopy Methods 0.000 description 40
- 238000012360 testing method Methods 0.000 description 38
- 108020004414 DNA Proteins 0.000 description 36
- 238000006243 chemical reaction Methods 0.000 description 31
- 239000000047 product Substances 0.000 description 31
- 239000007788 liquid Substances 0.000 description 23
- 102000053602 DNA Human genes 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
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- 238000004458 analytical method Methods 0.000 description 11
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- 238000007846 asymmetric PCR Methods 0.000 description 10
- 239000011521 glass Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000001502 gel electrophoresis Methods 0.000 description 7
- 238000011002 quantification Methods 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 description 6
- 238000004364 calculation method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
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- 102000004190 Enzymes Human genes 0.000 description 3
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- QTANTQQOYSUMLC-UHFFFAOYSA-O Ethidium cation Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 QTANTQQOYSUMLC-UHFFFAOYSA-O 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- CPBQJMYROZQQJC-UHFFFAOYSA-N helium neon Chemical compound [He].[Ne] CPBQJMYROZQQJC-UHFFFAOYSA-N 0.000 description 2
- 239000000138 intercalating agent Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ACNUVXZPCIABEX-UHFFFAOYSA-N 3',6'-diaminospiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(N)C=C1OC1=CC(N)=CC=C21 ACNUVXZPCIABEX-UHFFFAOYSA-N 0.000 description 1
- 230000005653 Brownian motion process Effects 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 206010051723 Fluctuance Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010056740 Genital discharge Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- ULHRKLSNHXXJLO-UHFFFAOYSA-L Yo-Pro-1 Chemical compound [I-].[I-].C1=CC=C2C(C=C3N(C4=CC=CC=C4O3)C)=CC=[N+](CCC[N+](C)(C)C)C2=C1 ULHRKLSNHXXJLO-UHFFFAOYSA-L 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- WZSUOQDIYKMPMT-UHFFFAOYSA-N argon krypton Chemical compound [Ar].[Kr] WZSUOQDIYKMPMT-UHFFFAOYSA-N 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 238000005537 brownian motion Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- UIZLQMLDSWKZGC-UHFFFAOYSA-N cadmium helium Chemical compound [He].[Cd] UIZLQMLDSWKZGC-UHFFFAOYSA-N 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000005315 distribution function Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000011842 forensic investigation Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000007850 in situ PCR Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 238000001579 optical reflectometry Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- ACOJCCLIDPZYJC-UHFFFAOYSA-M thiazole orange Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1.C1=CC=C2C(C=C3N(C4=CC=CC=C4S3)C)=CC=[N+](C)C2=C1 ACOJCCLIDPZYJC-UHFFFAOYSA-M 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- CPYIZQLXMGRKSW-UHFFFAOYSA-N zinc;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+3].[Fe+3].[Zn+2] CPYIZQLXMGRKSW-UHFFFAOYSA-N 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/60—Detection means characterised by use of a special device
- C12Q2565/601—Detection means characterised by use of a special device being a microscope, e.g. atomic force microscopy [AFM]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30131698A JP4262336B2 (ja) | 1998-10-22 | 1998-10-22 | 標的核酸の定量分析方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE19950823A1 true DE19950823A1 (de) | 2000-04-27 |
Family
ID=17895396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE1999150823 Withdrawn DE19950823A1 (de) | 1998-10-22 | 1999-10-21 | Quantitatives Verfahren zum Analysieren einer Targetnukleinsäure |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP4262336B2 (https=) |
| DE (1) | DE19950823A1 (https=) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3910000B2 (ja) * | 2000-03-27 | 2007-04-25 | オリンパス株式会社 | １塩基置換検出方法 |
| JP2001269198A (ja) * | 2000-03-27 | 2001-10-02 | Olympus Optical Co Ltd | 多型遺伝子の型を決定する方法 |
| EP1180549B1 (en) * | 2000-03-27 | 2007-02-21 | Olympus Corporation | Method of measuring polymorphism |
| JP4749622B2 (ja) * | 2001-08-10 | 2011-08-17 | オリンパス株式会社 | Ｄｎａエンドヌクレアーゼ活性の検出法 |
| JP2003177131A (ja) * | 2001-12-11 | 2003-06-27 | Olympus Optical Co Ltd | 生物学的な結合親和性を検出する方法 |
| JP2010178716A (ja) * | 2009-02-09 | 2010-08-19 | Olympus Corp | 標的核酸分子の定量方法及び標的核酸分子定量キット |
| JP6921638B2 (ja) | 2017-06-16 | 2021-08-18 | 株式会社東芝 | 核酸検出定量方法、チップ、アッセイキット、核酸検出定量装置及びプログラム |
-
1998
- 1998-10-22 JP JP30131698A patent/JP4262336B2/ja not_active Expired - Fee Related
-
1999
- 1999-10-21 DE DE1999150823 patent/DE19950823A1/de not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000125900A (ja) | 2000-05-09 |
| JP4262336B2 (ja) | 2009-05-13 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 8127 | New person/name/address of the applicant |
Owner name: OLYMPUS OPTICAL CO., LTD., TOKIO/TOKYO, JP |
|
| 8181 | Inventor (new situation) |
Free format text: KINJO, MASATAKA, SAPPORO, JP |
|
| 8110 | Request for examination paragraph 44 | ||
| 8139 | Disposal/non-payment of the annual fee |