DK1910572T3 - Fremgangsmåder til detektering af nukleinsyrer i enkelte celler og til identificering af sjældne celler fra store heterogene cellepopulationer - Google Patents

Fremgangsmåder til detektering af nukleinsyrer i enkelte celler og til identificering af sjældne celler fra store heterogene cellepopulationer Download PDF

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DK1910572T3
DK1910572T3 DK06785111.3T DK06785111T DK1910572T3 DK 1910572 T3 DK1910572 T3 DK 1910572T3 DK 06785111 T DK06785111 T DK 06785111T DK 1910572 T3 DK1910572 T3 DK 1910572T3
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nucleic acid
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Yuling Luo
Shiping Chen
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Advanced Cell Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • 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
    • C12Q1/682—Signal amplification
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    • 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
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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Claims (17)

1. Fremgangsmåde til detektering af et eller flere nukleinsyretargets i en enkelt celle, hvilken fremgangsmåde omfatter: at tilvejebringe en prøve, der omfatter cellen, hvilken celle omfatter eller er mistænkt for at omfatte et eller flere nukleinsyretargets; at fiksere og permeabilisere cellen; for hvert nukleinsyretarget at tilvejebringe (i) en eller flere markeringssonder, hvor hver markeringssonde omfatter en eller flere markeringer, (ii) en eller flere markeringssonder og en eller flere forstærkere, hvor hver markeringssonde omfatter en eller flere markeringer, og hvor hver forstærker kan hybridisere til en eller flere markeringssonder, eller (iii) en eller flere markeringssonder, en eller flere forstærkere og en eller flere forforstærkere, hvor hver markeringssonde omfatter en eller flere markeringer, hvor hver forstærker kan hybridisere til en eller flere markeringssonder, og hvor hver forforstærker kan hybridisere til en eller flere forstærkere; for hvert nukleinsyretarget at tilvejebringe to eller flere forskellige indfangningssonder, hvor hver af de to eller flere indfangningssonder omfatter et T-afsnit, der er komplementært med et afsnit på omtalte nukleinsyretarget, og et L-afsnit, der er komplementært med et afsnit på markeringssonden, eller på en forstærker eller på en forforstærker, og hvor T-afsnittene er komplementære med ikke-overlappende regioner af omtalte nukleinsyretarget, og L-afsnittene er komplementære med ikke-overlappende regioner af markeringssonden, hvor forstærkeren eller forforstærkeren; at hybridisere, i cellen, de to eller flere indfangningssonder til en enkelt kopi af omtalte nukleinsyretarget ved tilstedeværelse i cellen; at indfange markeringssonden til de to eller flere indfangningssonder, hvorved markeringssonden til omtalte nukleinsyretarget indfanges, ved samtidig hybridisering af mindst to forskellige indfangningssonder til en enkelt kopi af markeringssonden, eller ved samtidig hybridisering af mindst to forskellige indfangningssonder til en enkelt kopi af forstærkeren og hybridisering af markeringssonderne til forstærkeren, eller ved samtidig hybridisering af mindst to forskellige indfangningssonder til en enkelt kopi af forforstærkeren og hybridisering af den ene eller flere forstærkere til forforstærkeren og den ene eller flere markeringssonder til hver af den ene eller flere forstærkere; og at detektere et signal fra markeringen.
2. Fremgangsmåde ifølge krav 1, hvor de to eller flere forskellige indfangningssonder hybridiserer til unikke og naboliggende afsnit på omtalte nukle-insyretarget.
3. Fremgangsmåde ifølge krav 1, hvor hybridisering af de to eller flere forskellige indfangningssonder til den enkelte kopi af markeringssonden, forstærkeren eller forforstærkeren udføres ved en hybridiseringstemperatur, der er højere end smeltetemperaturen Tm af L-afsnittet af hver enkelt indfangningssonde, der binder til markeringssonden, forstærkeren eller forforstærkeren.
4. Fremgangsmåde ifølge krav 3, hvor hybridisering af de to eller flere forskellige indfangningssonder til den enkelte kopi af omtalte nukleinsyretarget udføres ved en hybridiseringstemperatur, der er lavere end smeltetemperaturen Tm af T-afsnittet af hver enkelt indfangningssonde, der binder til omtalte nukleinsyretarget.
5. Fremgangsmåde ifølge krav 1, hvor hybridisering af de to eller flere indfangningssonder til den enkelte kopi af omtalte nukleinsyretarget udføres ved en hybridiseringstemperatur, der er højere end smeltetemperaturen Tm af T-afsnittet af hver enkelt indfangningssonde, der binder til omtalte nukleinsyretarget.
6. Fremgangsmåde ifølge krav 5, hvor hybridisering af de to eller flere forskellige indfangningssonder til den enkelte kopi af markeringssonden, forstærkeren eller forforstærkeren udføres ved en hybridiseringstemperatur, der er lavere end smeltetemperaturen Tm af L-afsnittet af hver enkelt indfangningssonde, der binder til markeringssonden, forstærkeren eller forforstærkeren.
7. Fremgangsmåde ifølge krav 1, endvidere omfattende at tilvejebringe en eller flere blokeringssonder, der kan hybridisere til regioner af omtalte nukleinsyretarget, der ikke er optaget af indfangningssonderne.
8. Fremgangsmåde ifølge krav 1, hvor hvert hybridiserings- eller indfangningstrin udføres for alle nukleinsyretargets samtidigt.
9. Fremgangsmåde ifølge krav 1, hvor et eller flere nukleinsyretargets uafhængigt af hinanden vælges fra gruppen bestående af et DNA, et kromosomalt DNA, et RNA, et mRNA, et microRNA, et ribosomalt RNA, en nukleinsyre, der er endogen for cellen, og en nukleinsyre, der er indført i eller ekspri-meret i cellen ved infektion af cellen med et patogen.
10. Fremgangsmåde ifølge krav 1, hvor det ene eller flere nukleinsyretargets omfatter et første nukleinsyretarget, der omfatter en første region af et mRNA og et andet nukleinsyretarget, der omfatter en anden region af samme mRNA.
11. Fremgangsmåde ifølge krav 1, hvor det ene eller flere nukleinsyretargets omfatter en referencenukleinsyre, og hvor fremgangsmåden omfatter at normalisere signalet af det ene eller flere nukleinsyretargets til signalet af refe-rencenukleinsyren.
12. Fremgangsmåde ifølge krav 1, endvidere omfattende trinnet med at korrelere intensiteten af signalet af hvert nukleinsyretarget med en mængde af det tilsvarende nukleinsyretarget, der er til stede i cellen.
13. Fremgangsmåde ifølge krav 1, hvor prøven, der omfatter cellen, stammer fra en kropsvæske eller fra blod.
14. Fremgangsmåde ifølge krav 1, hvor prøven omfatter et vævsafsnit.
15. Fremgangsmåde ifølge krav 1, hvor cellen er i suspension under hybridiserings-, indfangnings- og/eller detekteringstrinnene.
16. Fremgangsmåde ifølge krav 1, hvor cellen er en cirkulerende tumorcelle.
17. Fremgangsmåde ifølge krav 1, hvor hver indfangningssonde er i en "Z"-udformning.
DK06785111.3T 2005-06-20 2006-06-19 Fremgangsmåder til detektering af nukleinsyrer i enkelte celler og til identificering af sjældne celler fra store heterogene cellepopulationer DK1910572T3 (da)

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PCT/US2006/023816 WO2007001986A2 (en) 2005-06-20 2006-06-19 Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations

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DK06785111.3T DK1910572T3 (da) 2005-06-20 2006-06-19 Fremgangsmåder til detektering af nukleinsyrer i enkelte celler og til identificering af sjældne celler fra store heterogene cellepopulationer

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