DK2500436T3 - Fremgangsmåde, sonde og kit til in situ-hybridisering af DNA og anvendelse deraf - Google Patents
Fremgangsmåde, sonde og kit til in situ-hybridisering af DNA og anvendelse deraf Download PDFInfo
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- DK2500436T3 DK2500436T3 DK11290139.2T DK11290139T DK2500436T3 DK 2500436 T3 DK2500436 T3 DK 2500436T3 DK 11290139 T DK11290139 T DK 11290139T DK 2500436 T3 DK2500436 T3 DK 2500436T3
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Claims (16)
1. Fremgangsmåde til både detektering af forekomst af initiering af replikati-onshændelser i genomisk DNA, som er mitokondrielt genomisk DNA, og detektering af mitokondrielt DNA-transkription i en eukaryotisk celle, omfattende trinnene: - at bringe den eukaryotiske celle omfattende mitokondrielt genomisk DNA i kontakt med en første nukleotidsonde, under betingelser, der muliggør in si-fry-hybridisering af den første nukleotidsonde med en målregion i det mito-kondrielle DNA-genom, hvor målregionen omfatter en nukleinsyresekvens, som ikke har nogen identificeret tilsvarende annealing RNA i en metabolisk aktiv celle og derfor forbliver RNA-fri under transkription og replikation af det mitokondrielle DNA-genom, hvilken første nukleotidsonde er specifik for et segment af ikke-transkriberet mitokondrielt gDNA og omfatter eller består af: 1. nukleinsyren med sekvensen med et hvilket som helst af SEQ ID N°1 til SEQ ID N°16 eller et komplement deraf eller ii. en nukleinsyre, der har mindst 80 % identitet med nukleinsyresekvensen med et hvilket som helst af SEQ ID N°1 til SEQ ID N°16 eller et komplement deraf, hvilket nukleinsyremolekyle er enten et enkeltstrenget molekyle eller et dobbeltstrenget molekyle, og - at bringe den eukaryotiske celle i kontakt med mindst en anden nukleotidsonde, der er målrettet mindst et mitokondrielt RNA-molekyle svarende til en transkriberet region af et mitokondrielt DNA-molekyle i cellen, og - at detektere den første nukleotidsonde, der er hybridiseret til det mitokondrielle DNA, og den anden nukleotidsonde, der er hybridiseret til det mitokondrielle RNA-molekyle.
2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at den omfatter trinnet med delvis denaturering af det genomiske DNA-molekyle omfattende målregionen ved hjælp af et af følgende trin: i. ved hjælp af opvarmning af den eukaryotiske celle omfattende det genomiske DNA ved en temperatur i området 72 til 78 °C, fortrinsvis 75 °C, i 2 til 8 minutter, fortrinsvis 4 til 5 minutter, især 5 minutter, ii. eller i nærvær af et kemisk middel, såsom formamid, f.eks. en opløsning af 70 % formamid, eller ved hjælp af begge trin.
3. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 2, kendetegnet ved, at den første nukleotidsonde er et enkeltstrenget DNA-fragment, hvis størrelse varierer fra 80 bp til 3000 bp, eller fra 90 til 150 bp, især fra 95 til 110 bp, fortrinsvis med en størrelse på 99 bp.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 3, kendetegnet ved, at den første nukleotidsonde er målrettet den mitokondrielle genomiske DNA-sekvens, der ligger mellem de to promotore PH1 (HSP) og LSP af det mitokondrielle genom af en eukaryotisk celle, eller er komplementær til en nukleinsyresekvens, der har mindst 80 % identitet med den mål-DNA-region, der ligger mellem de to promotore PH1 (HSP) og LSP af det mitokondrielle genom af en eukaryotisk celle.
5. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 4, kendetegnet ved, at den første nukleotidsonde, og eventuelt den anden nukleotidsonde, er direkte markeret med en fluorescerende gruppe og/eller omfatter modificerede nukleotider.
6. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 5, kendetegnet ved, at detekteringen af nukleinsyresekvensen af målregionen på det mitokondrielle DNA-genom og det mitokondrielle RNA-molekyle opnås i ét trin, især samtidigt.
7. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 6, kendetegnet ved, at den udføres på (en) fikseret/fikserede celle(r) eller væv.
8. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 7, til in situ-hybridisering og detektering af mitokondrielt nukleotidmateriale inden for mindst én eukaryotisk celle, omfattende trinnene: a. At fiksere cellen i 1 til 4 % paraformaldehyd (PFA), fortrinsvis 2 % PFA i ca. 20 til 30 minutter, især 30 minutter, b. At permeabilisere den fikserede celle med 0,5 % til 1 % Triton X100 i PBS 1X, i ca. 5 til 10 minutter ved 4 °C, især 5 minutter, c. At denaturere nukleinsyreindholdet af den permeabiliserede fikserede celle ved hjælp af opvarmning ved en temperatur i et område på 72 til 78 °C, fortrinsvis 75 °C, i 2 til 8 minutter, fortrinsvis 4 til 5 minutter, især 5 minutter, d. At bringe nukleinsyren/nukleinsyrerne i cellen behandlet i henhold til trin (c) i kontakt med mitokondriel(le) nukleotidsonde(r) til muliggørelse af hybri-disering af mitokondriel(le) nukleinsyre(r) med sonden/sonderne, hvor son-den/sonderne har en størrelse i området fra 80 til 3000 nukleotider, eller fra 90 til 1000 nukleotider, især fra 95 til 110 nukleotider, hvilke(n) nukleotidson-de(r) er indeholdt i en hybridiseringsopløsning omfattende fra 100 ng/μΙ til 10 pg/μΙ laksesperm-DNA, e. At detektere nukleinsyren/nukleinsyrerne hybridiseret til sonden/sonderne tilsat i trin (d).
9. Fremgangsmåde ifølge krav 8, yderligere omfattende et trin med at vaske den celle, der er bragt i kontakt med den/de mitokondrielle nukleotidsonde(r), med en egnet buffer inden detekteringstrinnet.
10. Fremgangsmåde ifølge et hvilket som helst af kravene 8 eller 9, hvor hy-bridiseringstrinnet udføres i løbet af ca. 15 timer ved 37 °C.
11. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 10, yderligere omfattende trin, der muliggør markering og detektering af mindst et protein af interesse i den eukaryotiske celle, hvor detekteringen af mitokondrielt nu-kleotidmateriale og af proteinet af interesse eventuelt opnås i ét trin, især samtidigt.
12. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 11, yderligere omfattende et trin med analyse af resultatet/resultaterne af den/de udførte detektering(er).
13. Nukleinsyremolekyle, der hybridiserer med en målregion i et eukaryotisk mitokondrielt genomisk DNA, hvor målregionen omfatter en nukleinsyrese-kvens, der ikke har nogen identificeret tilsvarende annealing RNA i den metabolisk aktive celle indeholdende det eukaryotiske mitokondrielle genomiske DNA og derfor forbliver RNA-fri under transkription og replikation af det mito-kondriele DNA-genom, hvor nukleinsyremolekylet er specifikt for et segment af ikke-transkriberet mitokondrielt gDNA, bestående af: i. nukleinsyren med sekvensen med et hvilket som helst af SEQ ID N°1 til SEQ ID N°16 eller et komplement deraf eller ii. en nukleinsyre, der har mindst 80 % identitet med nukleinsyresekvensen med et hvilket som helst af SEQ ID N°1 til SEQ ID N°16 eller et komplement deraf, hvilket nukleinsyremolekyle er enten et enkeltstrenget molekyle eller et dobbeltstrenget molekyle.
14. Nukleinsyremolekyle ifølge krav 13, som er markeret.
15. Kit til udførelse af in s/Yu-hybridisering på fikserede celler, omfattende en første nukleotidsonde bestående af et detekterbart nukleinsyremolekyle ifølge krav 13 eller krav 14 og omfattende en anden nukleotidsonde, der hybri-diserer med et mitokondrielt RNA-molekyle.
16. Anvendelse af fremgangsmåden, nukleinsyremolekylet eller kittet ifølge et hvilket som helst af kravene 1 til 15 til detektering af mitokondriesyg-dom(me) eller til detektering af neoplastisk(e) sygdom(me) eller cancer(e) eller til test af cytotoksiciteten af organiske eller kemiske forbindelser, især lægemidler, på eukaryotiske celler.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP11290139.2A EP2500436B1 (en) | 2011-03-17 | 2011-03-17 | Method, probe and kit for DNA in situ hybridation and use thereof |
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| DK2500436T3 true DK2500436T3 (da) | 2016-08-29 |
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| DK11290139.2T DK2500436T3 (da) | 2011-03-17 | 2011-03-17 | Fremgangsmåde, sonde og kit til in situ-hybridisering af DNA og anvendelse deraf |
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| Country | Link |
|---|---|
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| EP (2) | EP2500436B1 (da) |
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| EP2766498B1 (en) | 2011-10-14 | 2019-06-19 | President and Fellows of Harvard College | Sequencing by structure assembly |
| EP4108782B1 (en) | 2011-12-22 | 2023-06-07 | President and Fellows of Harvard College | Compositions and methods for analyte detection |
| ES2991004T3 (es) | 2011-12-22 | 2024-12-02 | Harvard College | Métodos para la detección de analitos |
| US9914967B2 (en) | 2012-06-05 | 2018-03-13 | President And Fellows Of Harvard College | Spatial sequencing of nucleic acids using DNA origami probes |
| EP3578666A1 (en) | 2013-03-12 | 2019-12-11 | President and Fellows of Harvard College | Method of generating a three-dimensional nucleic acid containing matrix |
| SG10201710030QA (en) | 2013-06-04 | 2018-01-30 | Harvard College | Rna-guided transcriptional regulation |
| WO2016007839A1 (en) | 2014-07-11 | 2016-01-14 | President And Fellows Of Harvard College | Methods for high-throughput labelling and detection of biological features in situ using microscopy |
| CN108474022A (zh) | 2015-11-03 | 2018-08-31 | 哈佛学院董事及会员团体 | 用于包含三维核酸的基质容积成像的设备和方法 |
| EP3871695A1 (en) | 2015-12-07 | 2021-09-01 | Arc Bio, LLC | Methods and compositions for the making and using of guide nucleic acids |
| CA3022290A1 (en) | 2016-04-25 | 2017-11-02 | President And Fellows Of Harvard College | Hybridization chain reaction methods for in situ molecular detection |
| CN109983125B (zh) | 2016-08-31 | 2024-06-04 | 哈佛学院董事及会员团体 | 生成用于通过荧光原位测序检测的核酸序列文库的方法 |
| CN118853848A (zh) | 2016-08-31 | 2024-10-29 | 哈佛学院董事及会员团体 | 将生物分子的检测组合到使用荧光原位测序的单个试验的方法 |
| CN109789167A (zh) | 2017-04-14 | 2019-05-21 | 哈佛学院董事及会员团体 | 用于产生细胞衍生的微丝网络的方法 |
| FI3684949T3 (fi) | 2017-09-22 | 2024-07-17 | Univ Washington | Solumolekyylien kombinatorinen in situ -leimaaminen |
| WO2020028194A1 (en) | 2018-07-30 | 2020-02-06 | Readcoor, Inc. | Methods and systems for sample processing or analysis |
| WO2020076976A1 (en) | 2018-10-10 | 2020-04-16 | Readcoor, Inc. | Three-dimensional spatial molecular indexing |
| US20220049303A1 (en) | 2020-08-17 | 2022-02-17 | Readcoor, Llc | Methods and systems for spatial mapping of genetic variants |
| CN114891861B (zh) * | 2022-05-05 | 2024-07-26 | 江苏省食品药品监督检验研究院 | 一种检测抗体中cho细胞残留dna的raa试剂盒及其检测方法 |
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| US5541308A (en) * | 1986-11-24 | 1996-07-30 | Gen-Probe Incorporated | Nucleic acid probes for detection and/or quantitation of non-viral organisms |
| US6462190B1 (en) * | 1999-04-14 | 2002-10-08 | California Institute Of Technology | Polynucleotides and kits for detection of an age-related mutation |
| US20070190534A1 (en) | 2001-06-11 | 2007-08-16 | Genesis Genomics Inc. | Mitochondrial sites and genes associated with prostate cancer |
| USH2220H1 (en) * | 2001-08-10 | 2008-07-01 | Snp Consortium | Identification and mapping of single nucleotide polymorphisms in the human genome |
| WO2005003766A2 (en) * | 2003-06-13 | 2005-01-13 | Whitehead Institute For Biomedical Research | Methods of regulating metabolism and mitochondrial function |
| JP2005107404A (ja) | 2003-10-01 | 2005-04-21 | Matsushita Electric Ind Co Ltd | 広角撮像光学系、並びにそれを備えた広角撮像装置、監視用撮像装置、車載用撮像装置及び投写装置 |
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| US20140087378A1 (en) | 2014-03-27 |
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| EP2500436B1 (en) | 2016-05-25 |
| US11566280B2 (en) | 2023-01-31 |
| EP2686440A1 (en) | 2014-01-22 |
| JP2014509862A (ja) | 2014-04-24 |
| JP6262533B2 (ja) | 2018-01-17 |
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