EP1841886A2 - Procede d'hybridation avec des acides nucleiques - Google Patents
Procede d'hybridation avec des acides nucleiquesInfo
- Publication number
- EP1841886A2 EP1841886A2 EP06705849A EP06705849A EP1841886A2 EP 1841886 A2 EP1841886 A2 EP 1841886A2 EP 06705849 A EP06705849 A EP 06705849A EP 06705849 A EP06705849 A EP 06705849A EP 1841886 A2 EP1841886 A2 EP 1841886A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- hybridization
- microwave
- nucleic acid
- hybridisation
- nucleic acids
- 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- Withdrawn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
- 238000009396 hybridization Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 19
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 11
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 11
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000007899 nucleic acid hybridization Methods 0.000 claims description 7
- 239000013611 chromosomal DNA Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 13
- 238000004904 shortening Methods 0.000 abstract 1
- 238000007901 in situ hybridization Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000031864 metaphase Effects 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000020169 heat generation Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000012775 microarray technology Methods 0.000 description 1
- 238000007431 microscopic evaluation Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000005406 washing Methods 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
- C12Q1/6832—Enhancement of hybridisation reaction
Definitions
- the invention relates to a method for rapid in situ hybridization with nucleic acids, for example for the nucleic acid samples to be evaluated.
- Fields of application for this are on the one hand in-situ hybridizations which are under time pressure, such as. In prenatal diagnostics or hard to hybridize or small nucleic acid probes which hybridize more reliably by the method.
- DNA sequences in cell nuclei, in chromosomes or in general in tissues and tissue sections, but also already bound to special surfaces, such as glass in conjunction with array techniques overview eg., Forster T, Roy D, Ghazal P: Experiments using microarray technology: limitations and standard operating procedures, J. Endocrinol., 178, 2003, 195-204).
- the target sequences are often difficult to obtain for the sample sequences (for example DNA sequences in chromosomal structures, Saitoh Y, Laemmli UK: Metaphase
- chromosome strueture bands arise from a differential folding path of the highly AT-rich scaffold, Cell 76, 1994, 609-622).
- the melting of the target sequences (denaturation of, for example, the DNA) is achieved before the hybridization, in particular by heat.
- Microwaves have heretofore been used only for hybridization enhancement on tissue sections in immunohistochemistry (eg Coates PJ, Hall PA, Butler MG, DArdenne AJ: Rapid technique of DNA-DNA in situ hybridization on formalin fixed tissue sections using microwave irradiation, J Clin Pathol 40, 1987, 865-869: Bull JH, Harnden P: Efficient nuclear FISH on paraffin-embedded tissue sections using microwave pretreatment, Biotechniques 26, 1999, 416-418, 422, Kobayashi K, Kitayama Y, Igarashi H, Yoshino G, Kobayashi T, Kazui T, Sugimura H: Intratumor heterogeneity of centromeric numerical abnormality in multiple primary gastric cancers: application of fluorescence in situ hybridization with intermittent microwave irradiation on paraffin-embedded tissue, Jpn J Cancer Res 91, 2000, 1134- 1141).
- microwaves were used on chromosomal DNA as well as tissue sections just prior to the hybridization process in order to transfer the target sequences (via a thermal effect) from the double stranded form into the single stranded form (melting of the double strand) and thus for hybridization with sample DNA first to make accessible (eg Ko E 5 Rademaker A 5 Martin R: Microwave decondensation and codenaturation: a new methodology to maximize FISH data from donors with very low concentrations of sperm, Cytogenet Cell Genet 95, 2001, 143-145).
- This effect affects the usefulness of the target hybridization sequences per se, but not the efficiency of subsequent hybridization.
- the most time-consuming step is by far step 4, which takes two to four hours (centromere probes) or one to several days (single copy and very small probes) depending on the type of probe.
- the invention is therefore based on the object to improve the in situ hybridization with nucleic acids and to shorten the hybridization process.
- the method for nucleic acid hybridization is carried out under the influence of microwaves.
- the hybridization results qualitatively leave a much better impression, mediated by more specific hybridization signals. This is particularly important in the use of small and single-copy nucleic acid probes, whose conventional hybridization often leads to background-rich non-evaluable results.
- the microwave effect according to the invention is very largely independent of the heat generation at the target sequences on the hybridization process itself. It is rather to start from a microwave-mediated excitation of the molecular movements, which sterically facilitates and improves the achievement of the target sequences by the sample sequences.
- the nucleic acid hybridization under the influence of microwaves preferably takes place at a hybridization temperature of at most 37 ° C.
- the hybridization temperature can advantageously be regulated by a cold water bath by determining the microwave exposure time on the basis of calibration curves corresponding to the water temperature and the water volume of the cold water bath such that at the end the microwave exposure time reaches a maximum water temperature of 37 ° C.
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)
Abstract
L'objet de la présente invention est d'améliorer l'hybridation in situ avec des acides nucléiques et de raccourcir le processus d'hybridation. A cet effet, ledit procédé d'hybridation d'acides nucléiques est effectué sous l'influence de micro-ondes à une température d'hybridation maximale de 37 °C. La présente invention est utilisée par exemple pour marquer et évaluer des sondes d'acide nucléique.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102005003638A DE102005003638A1 (de) | 2005-01-24 | 2005-01-24 | Verfahren zur Nukleinsäurehybridisierung |
| PCT/DE2006/000113 WO2006076912A2 (fr) | 2005-01-24 | 2006-01-24 | Procédé d'hybridation avec des acides nucléiques |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1841886A2 true EP1841886A2 (fr) | 2007-10-10 |
Family
ID=36406052
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP06705849A Withdrawn EP1841886A2 (fr) | 2005-01-24 | 2006-01-24 | Procede d'hybridation avec des acides nucleiques |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US20080108805A1 (fr) |
| EP (1) | EP1841886A2 (fr) |
| DE (1) | DE102005003638A1 (fr) |
| WO (1) | WO2006076912A2 (fr) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2187289B1 (es) * | 2001-10-02 | 2004-10-16 | Consulting, Comunicacio I Disseny, S.L. | Telefono movil de comunicacion personal. |
-
2005
- 2005-01-24 DE DE102005003638A patent/DE102005003638A1/de not_active Withdrawn
-
2006
- 2006-01-24 US US11/795,996 patent/US20080108805A1/en not_active Abandoned
- 2006-01-24 WO PCT/DE2006/000113 patent/WO2006076912A2/fr not_active Ceased
- 2006-01-24 EP EP06705849A patent/EP1841886A2/fr not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO2006076912A2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2006076912A3 (fr) | 2006-09-08 |
| US20080108805A1 (en) | 2008-05-08 |
| WO2006076912A2 (fr) | 2006-07-27 |
| DE102005003638A1 (de) | 2006-07-27 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 20070726 |
|
| AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR |
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| RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: WEISE, ANJA Inventor name: CLAUSSEN, UWE Inventor name: LIEHR, THOMAS Inventor name: HALBHUBER, KARL-JUERGEN |
|
| 17Q | First examination report despatched |
Effective date: 20071116 |
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| DAX | Request for extension of the european patent (deleted) | ||
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20090801 |