NO175380B - Method for Quantitative Determination of Nucleic Acid Molecules and Reagent Packages for Use in Performing the Process - Google Patents
Method for Quantitative Determination of Nucleic Acid Molecules and Reagent Packages for Use in Performing the ProcessInfo
- Publication number
- NO175380B NO175380B NO870794A NO870794A NO175380B NO 175380 B NO175380 B NO 175380B NO 870794 A NO870794 A NO 870794A NO 870794 A NO870794 A NO 870794A NO 175380 B NO175380 B NO 175380B
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- Prior art keywords
- fragment
- gene
- hindiii
- nucleic acid
- standard
- Prior art date
Links
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Classifications
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- 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
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- 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
- C12Q1/6876—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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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Description
Oppfinnelsen angår en fremgangsmåte til kvantitativ bestemmelse av visse nukleinsyremolekyler, særlig graden av amplifikasjon av gener og/eller tilsvarende budbringer RNA-molekyler ved bruk av sandwich- eller oppløsnings-hybridiseringsmetoden. En annen side ved oppfinnelsen angår den reagenspakke som anvendes ved fremgangsmåten. The invention relates to a method for the quantitative determination of certain nucleic acid molecules, in particular the degree of amplification of genes and/or corresponding messenger RNA molecules using the sandwich or solution hybridization method. Another aspect of the invention relates to the reagent pack used in the method.
Antallet kopier individuelle gener i genomet er vanligvis konstant. I noen tilfeller er der bare ett gen pr. haploid genom, og i andre tilfeller er der flere. Under visse omstendig-heter kan antallet kopier forandre seg. Amplifikasjonen av visse gener er f.eks. funnet å ha forbindelse med utviklingen av kreft. Det er også vist at ytre faktorer, såsom farmasøytika og metaller etc. forårsaker amplifikasjon av visse gener. For utviklingen av en sykdom er det feilaktige eller økede ekspresjonsnivå av et gen såsom et onkogen, dvs. mengden av budbringer RNA i cellen, av største viktighet. Økede antall av noen kromosomer er årsaken til visse arvelige sykdommer eller andre forstyrrelser, mens noen arvelige sykdommer bare krever duplika-sjon av ett recessivt gen. I alle slike tilfeller er det viktig å bestemme antallet kromosomer eller gener som foreligger. The number of copies of individual genes in the genome is usually constant. In some cases, there is only one gene per haploid genome, and in other cases there are several. Under certain circumstances, the number of copies may change. The amplification of certain genes is e.g. found to be associated with the development of cancer. It has also been shown that external factors such as pharmaceuticals and metals etc. cause amplification of certain genes. For the development of a disease, the incorrect or increased expression level of a gene such as an oncogene, i.e. the amount of messenger RNA in the cell, is of utmost importance. Increased numbers of some chromosomes are the cause of certain hereditary diseases or other disorders, while some hereditary diseases only require the duplication of one recessive gene. In all such cases it is important to determine the number of chromosomes or genes present.
Antallet av visse DNA-molekyler, f.eks. graden av amplifikasjon av gitte gener, bestemmes for tiden ved digestering av det ekstraherte DNA som skal undersøkes ved hjelp av restriksjonsenzymer og ved separering av nukleotidfragmentene i henhold til lengde ved agarosegel-elektroforese. Deretter blir det enkelttrådede DNA overført og festet til et nitrocellulosefilter, hvor hybridisering finner sted ved anvendelse av det gen som skal undersøkes eller en del av dette gen som en sonde. Resultatene fås ved autoradiografi (Southern, J. Mol. Biol. 98, pp. 503-517, 1975). I hver parallell analyse er mengden av cellulært DNA den samme. The number of certain DNA molecules, e.g. the degree of amplification of given genes is currently determined by digesting the extracted DNA to be examined with restriction enzymes and by separating the nucleotide fragments according to length by agarose gel electrophoresis. The single-stranded DNA is then transferred and fixed to a nitrocellulose filter, where hybridization takes place using the gene to be investigated or part of this gene as a probe. The results are obtained by autoradiography (Southern, J. Mol. Biol. 98, pp. 503-517, 1975). In each parallel assay, the amount of cellular DNA is the same.
Intensitetene av hybridiseringsbåndene, dvs. signalene, sammenlignes og forholdene mellom kopitallene av genene under under-søkelse i forsøksprøvene anslås. Fremgangsmåten gir bare tilnærmede resultater. På samme måte måles RNA ved bruk av Northern-blotting-metoder eller dot-blotting-metoder. Disse metoder er kvantitativt meget unøyaktige (Thomas, Methods in Enzymol-, 100. pp. 255-266, 1983). The intensities of the hybridization bands, i.e. the signals, are compared and the ratios between the copy numbers of the genes under investigation in the experimental samples are estimated. The procedure only gives approximate results. Similarly, RNA is measured using Northern blotting methods or dot blotting methods. These methods are quantitatively very inaccurate (Thomas, Methods in Enzymol-, 100. pp. 255-266, 1983).
Kjente metoder, såsom Southern- og Northern-blottingsmetoder er langsomme og vanskelige å utføre. Da de bare gir tilnærmede resultater, er deres diagnostiske verdi tvilsom i tilfeller hvor det er viktig å kjenne antallet av visse nukleinsyremolekyler pr. gitt enhet, såsom en celle. Known methods, such as Southern and Northern blotting methods are slow and difficult to perform. As they only give approximate results, their diagnostic value is questionable in cases where it is important to know the number of certain nucleic acid molecules per given unit, such as a cell.
Sandwich- eller oppløsnings-hybridiseringsmetoder beskrevet i US-PS 4.486.539 og i britisk patentsøknad nr. GB 2 169 403 er kvantitative (Virtanen et al., Lancet 1, pp. 381-383, 1983). Dessuten krever fremgangsmåten ifølge oppfinnelsen en standard nukleinsyre, hvis kopitall er konstant og tillater bestemmelsen av antallet relevante nukleinsyremolekyler pr. gitt enhet, såsom en celle, nukleus, ribosom eller kromosom. Sandwich or solution hybridization methods described in US-PS 4,486,539 and in British Patent Application No. GB 2,169,403 are quantitative (Virtanen et al., Lancet 1, pp. 381-383, 1983). Moreover, the method according to the invention requires a standard nucleic acid, whose copy number is constant and allows the determination of the number of relevant nucleic acid molecules per given unit, such as a cell, nucleus, ribosome or chromosome.
Formålet med oppfinnelsen er å skaffe en nøyaktig og hurtig kvantitativ metode til nukleinsyremolekylbestemmelse som også er hurtigere og enklere å utføre enn de metoder som for tiden brukes. Den kan anvendes til diagnose av kreft og prenatal diagnose, for påvisning av agenter som bevirker genamplifikasjon og til demonstrasjon av utviklingen av f.eks. medikamentresistens, samt til bestemmelsen av ekspresjonsnivået av budbringer RNA. Oppfinnelsens formål oppnås ved en fremgangsmåte og en reagenspakke til bruk i fremgangsmåten, kjennetegnet ved de trekk som fremgår av patentkravene. The purpose of the invention is to provide an accurate and rapid quantitative method for nucleic acid molecular determination which is also faster and easier to perform than the methods currently used. It can be used for the diagnosis of cancer and prenatal diagnosis, for the detection of agents that cause gene amplification and for demonstration of the development of e.g. drug resistance, as well as to the determination of the expression level of messenger RNA. The purpose of the invention is achieved by a method and a reagent pack for use in the method, characterized by the features that appear in the patent claims.
Minst to bestemmelser er nødvendige i den foreliggende oppfin-nelse. Én bestemmer nukleinsyremolekylet, som kan foreligge i flere kopier, forsøksnukleinsyren. Den andre bestemmer det grunnleggende nukleinsyremolekyl som med fordel foreligger i konstant antall, standardnukleinsyren. I fremgangsmåten ifølge oppfinnelsen angir et nukleinsyremolekyl en bestemt nukleotidsekvens på 10-12 nukleotider eller et gen inneholdende flere tusen nukleotider. Det kan også bety et budbringer RNA eller en nukleotidsekvens betraktelig lengre enn et enkelt gen, dvs. et ampiikon. At least two provisions are necessary in the present invention. One determines the nucleic acid molecule, which may exist in multiple copies, the experimental nucleic acid. The second determines the basic nucleic acid molecule which is advantageously present in constant numbers, the standard nucleic acid. In the method according to the invention, a nucleic acid molecule indicates a specific nucleotide sequence of 10-12 nucleotides or a gene containing several thousand nucleotides. It can also mean a messenger RNA or a nucleotide sequence considerably longer than a single gene, i.e. an ampicon.
Bestemmelsen av forsøks- og standard-nukleinsyrer utføres ved bruk av en ellers vanlig sandwich-hybridiseringsmetode beskrevet f.eks. i US-PS 4.486.539 eller en oppløsnings-hybridiseringsmetode beskrevet i britisk patentsøknad nr. GB 2 169 403. Oppfinnelsen angår også en reagenspakke inneholdende nukleinsyrereagenser bestående av minst ett forsøkssondepar og minst ett standardsondepar. The determination of test and standard nucleic acids is carried out using an otherwise common sandwich hybridization method described e.g. in US-PS 4,486,539 or a solution hybridization method described in British patent application No. GB 2 169 403. The invention also relates to a reagent pack containing nucleic acid reagents consisting of at least one experimental probe pair and at least one standard probe pair.
Reagensene eller sondene som anvendes i fremgangsmåten, fremstilles ved bruk av rekombinant DNA-teknikker fra nukleinsyrer som er tilstrekkelig homologe med forsøks- og standardnuklein-syrene. Tilstrekkelig homologe nukleinsyrer kan også fremstilles syntetisk og semisyntetisk. The reagents or probes used in the method are prepared using recombinant DNA techniques from nucleic acids that are sufficiently homologous to the experimental and standard nucleic acids. Sufficiently homologous nucleic acids can also be produced synthetically and semisynthetically.
Forsøks- og standard-nukleinsyrene kan isoleres direkte fra celler og identifiseres ved forskjellige hybridiseringsteknik-ker. Slike forsøks- og standardnukleinsyrer er imidlertid også tilgjengelige i handelen og fra forskjellige genbanker. Forsøks-og standard-nukleinsyrer kan være enten DNA eller RNA. The experimental and standard nucleic acids can be isolated directly from cells and identified by various hybridization techniques. However, such experimental and standard nucleic acids are also available commercially and from various gene banks. Test and standard nucleic acids can be either DNA or RNA.
Sondepar som er egnet for sandwich- eller oppløsnings-hybridise-ringsmetoden fremstilles fra nukleinsyrer som er tilstrekkelig homologe med forsøks- og standard-nukleinsyrene ved rekombinant DNA-teknikker. De relevante nukleinsyrer digesteres ved egnede restriksjonsenzymer; minst to av de resulterende restriksjons-fragmenter som er anordnet forholdsvis nær hinannnen klones til minst to egnede vektorer. Et av fragmentene, detektorsonden, merkes med en egnet detekterbar markør, og den andre, oppfan-gingssonden, blir enten festet til en egnet bærer eller et stoff festes til denne, hvilket stoff tillater separasjon av den resulterende hybrid fra hybridiseringsblandingen ved hjelp av et annet stoff, såsom den komplementære komponent av et affinitetspar. Probe pairs suitable for the sandwich or solution hybridization method are prepared from nucleic acids sufficiently homologous to the test and standard nucleic acids by recombinant DNA techniques. The relevant nucleic acids are digested by suitable restriction enzymes; at least two of the resulting restriction fragments which are arranged relatively close to each other are cloned into at least two suitable vectors. One of the fragments, the detector probe, is labeled with a suitable detectable label, and the other, the capture probe, is either attached to a suitable support or a substance is attached thereto, which substance allows separation of the resulting hybrid from the hybridization mixture by means of another substance, such as the complementary component of an affinity pair.
Forsøks- og standard-sondeparene kan bygges sammen i egnede reagenspakker, hvor forsøks- og standard-sondeparene er begge DNA eller RNA eller forsøkssondeparet er DNA og standardsondeparet er RNA, eller omvendt. Den forutgående og ytterligere behandling av prøvene før hybridiseringen og hybridiserings-betingelsene bør derfor rette seg etter de sondepar som benyttes i forsøket. The test and standard probe pairs can be built together in suitable reagent packs, where the test and standard probe pairs are both DNA or RNA or the test probe pair is DNA and the standard probe pair is RNA, or vice versa. The prior and further treatment of the samples before the hybridization and the hybridization conditions should therefore follow the probe pairs used in the experiment.
Fremgangsmåten ifølge oppfinnelsen er særlig egnet til bestemmelse av antallet nukleinsyremolekyler direkte fra cellulære homogenater. Fremgangsmåten kan selvsagt også anvendes til bestemmelse av rensede eller rene nukleinsyrer. Før fremgangsmåten ifølge oppfinnelsen utføres, bør imidlertid den mest egnede forbehandling av nukleinsyreprøven velges. The method according to the invention is particularly suitable for determining the number of nucleic acid molecules directly from cellular homogenates. The method can of course also be used for the determination of purified or pure nucleic acids. However, before the method according to the invention is carried out, the most suitable pretreatment of the nucleic acid sample should be selected.
Det er mulig å utføre både DNA- og RNA-bestemmelser ved bruk av fremgangsmåten ifølge oppfinnelsen. Deoksyribonukleinsyrer denatureres for oppnåelse av enkelttråder, om nødvendig. Enkelttrådede budbringer RNA-molekyler som potensielt forstyrrer hybridiseringsforsøket kan hydrolyseres, f.eks. ved alkalisk koking. Prøven denatureres ikke i forbindelse med ribonuklein-syrebestemmelser, da den dobbelttrådede dioksyribonukleinsyre ikke virker forstyrrende inn på RNA-bestemmelsen. Det er selvsagt mulig å bryte opp DNA med deoksyribonuklease, eller forandre det kjemisk eller mekanisk slik at det ikke kan ta del i hybridiseringsreaksjonen. Derfor må, i forbindelse med DNA- og RNA-bestemmelser, en egnet fremgangsmåte til ytterligere behanding av prøven velges, eller alternativt denne ytterligere behandling kan sløyfes. Valget av en egnet fremgangsmåte for den videre behandling er selvsagt avhengig av fremgangsmåten som benyttes i forbehandlingen av nukleinsyreprøven. En rekke fremgangsmåter til forbehandling og videre behandling av nukleinsyreprøver er blitt beskrevet i litteraturen, hvilket tillater at den mest egnede metode kan velges i hvert tilfelle. It is possible to carry out both DNA and RNA determinations using the method according to the invention. Deoxyribonucleic acids are denatured to obtain single strands, if necessary. Single-stranded messenger RNA molecules that potentially interfere with the hybridization experiment can be hydrolyzed, e.g. by alkaline boiling. The sample is not denatured in connection with ribonucleic acid determinations, as the double-stranded dioxyribonucleic acid does not interfere with the RNA determination. It is of course possible to break up DNA with deoxyribonuclease, or to change it chemically or mechanically so that it cannot take part in the hybridization reaction. Therefore, in connection with DNA and RNA determinations, a suitable method for further processing of the sample must be selected, or alternatively this further processing can be omitted. The choice of a suitable method for the further processing obviously depends on the method used in the pre-processing of the nucleic acid sample. A number of methods for pre-treatment and further treatment of nucleic acid samples have been described in the literature, which allows the most suitable method to be selected in each case.
Bestemmelser hvor både forsøks- og standard-nukleinsyrene er enten DNA eller RNA kan utføres ved bruk av en udelt prøve. Bestemmelser hvor forsøksnukleinsyrene er DNA og standardnuk-leinsyrene RNA eller omvendt, må utføres ved bruk av en delt prøve, da forskjellige fremgangsmåter for videre behandling er nødvendige. Prøven kan selvsagt deles, selv om forsøks- og standard-nukleinsyrene er av den samme nukleinsyretype. Determinations where both the test and standard nucleic acids are either DNA or RNA can be performed using an undivided sample. Determinations where the experimental nucleic acids are DNA and the standard nucleic acids RNA or vice versa must be carried out using a split sample, as different methods for further processing are necessary. The sample can of course be split, even if the test and standard nucleic acids are of the same nucleic acid type.
Selve hybridiseringsforsøket utføres ved at den udelte prøve-oppløsning bringes i berøring samtidig med minst ett forsøks-sondepar og ett standardsondepar. Dersom prøveoppløsningen er blitt delt, bringes den separat i berøring med minst ett forsøkssondepar og et standardsondepar. I slike tilfeller blir mengden av forsøksnukleinsyre bestemt i én reaksjonsbeholder og mengden av standardnukleinsyre i den andre. The hybridization experiment itself is carried out by bringing the undivided sample solution into contact simultaneously with at least one experimental probe pair and one standard probe pair. If the sample solution has been divided, it is separately brought into contact with at least one experimental probe pair and a standard probe pair. In such cases, the amount of test nucleic acid is determined in one reaction container and the amount of standard nucleic acid in the other.
Uansett hvorvidt prøven deles eller ikke, tillates hybridiseringen å finne sted ved de mest fordelaktige betingelser og tidsrom i hvert tilfelle. Straks reaksjonen eller reaksjonene har funnet sted, blir de resulterende forsøks- og standardhybrider separert fra hybridiseringsblandingen eller -blandingene ved bæreren og vasket, eller ved et isolasjonsmiddel såsom det komplementære medlem av et affinitetspar. Markøren som er festet til forsøks-og standardhybridene måles og resultatet sammenlignes med standardkurver. På denne måte kan antallet nukleinsyremolekyler som skal undersøkes bestemmes pr. valgte enhet. Regardless of whether the sample is split or not, the hybridization is allowed to take place under the most favorable conditions and time period in each case. Once the reaction or reactions have taken place, the resulting test and standard hybrids are separated from the hybridization mixture or mixtures by the support and washed, or by an isolation agent such as the complementary member of an affinity pair. The marker attached to the experimental and standard hybrids is measured and the result compared with standard curves. In this way, the number of nucleic acid molecules to be examined can be determined per selected unit.
Fremgangsmåten ifølge oppfinnelsen er av praktisk diagnostisk verdi, særlig i påvisningen av visse typer kreft. I småcellede lungekarsinom er c-myc-genet ofte amplifisert og dets ekspresjonsnivå adskillig høyere enn i normalt vev. I tilfeller av neuroblastoma er N-myc-genet amplifisert. The method according to the invention is of practical diagnostic value, particularly in the detection of certain types of cancer. In small cell lung carcinoma, the c-myc gene is often amplified and its expression level is considerably higher than in normal tissue. In cases of neuroblastoma, the N-myc gene is amplified.
Fremgangsmåten ifølge oppfinnelsen kan også benyttes til å vise de mutagene eller karsinogene virkninger av visse midler eller utviklingen av medikamentresistens. Det er kjent at ytre seleksjonstrykk kan føre til øket ekspresjon av et bestemt gen. I behandlingen av kreft utvikler celler motstandsevne overfor et gitt medikament ved amplifikasjon av genet, hvis ekspresjonspro-dukt inaktiverer medikamentet. Et slikt tilfelle er metotreksat som induserer amplifikasjon av genet for dihydrofolatreduktase (DHFR). Et ytterligere eksempel er amplifikasjon av genet for metallotionin under påvirkning av kadmium. The method according to the invention can also be used to show the mutagenic or carcinogenic effects of certain agents or the development of drug resistance. It is known that external selection pressure can lead to increased expression of a particular gene. In the treatment of cancer, cells develop resistance to a given drug by amplification of the gene whose expression product inactivates the drug. One such case is methotrexate which induces amplification of the dihydrofolate reductase (DHFR) gene. A further example is the amplification of the gene for metallothionein under the influence of cadmium.
Ekspresjonsnivået for et gen er viktig fra fenotypen og celle-funksjonens synspunkt. Dette kan undersøkes ved måling av mengden budbringer RNA, som korrelerer med mengden protein som det koder for. Transkripsjonsproduktet av et onkogen bestemmer måten som dette til slutt vil uttrykkes på. The expression level of a gene is important from the point of view of phenotype and cell function. This can be investigated by measuring the amount of messenger RNA, which correlates with the amount of protein that it codes for. The transcription product of an oncogene determines the way in which it will ultimately be expressed.
Ekspresjonsnivåene av et onkogen varierer avhengig av celletype, differensieringsnivå og utviklingsfase av cellen. F.eks. på et visst stadium av fosterutvikling kopieres c-myc-onkogenet hurtig, mens på et annet trinn er denne meget langsom. Graden av amplifikasjon korrelerer ofte med ekspresjonsnivået av genet, skjønt det sistnevnte kan øke betydelig uten at den førstnevnte gjør det. I slike tilfeller bestemmes onkogenets rolle best ved måling av dets ekspresjonsnivå snarere enn antallet kopier. I noen tilfeller kan kvanititativ bestemmelse av budbringer RNA være enklere og mer praktisk enn kvantifisering av selve genproduktet. Som et eksempel kan nevnes c-myc-onkogenet, et labilt protein som lett koaguleres ved varme. The expression levels of an oncogene vary depending on the cell type, differentiation level and developmental phase of the cell. E.g. at a certain stage of fetal development the c-myc oncogene is copied rapidly, while at another stage this is very slow. The degree of amplification often correlates with the expression level of the gene, although the latter may increase significantly without the former. In such cases, the role of the oncogene is best determined by measuring its expression level rather than its copy number. In some cases, quantitative determination of messenger RNA may be easier and more practical than quantification of the gene product itself. As an example, the c-myc oncogene can be mentioned, a labile protein that is easily coagulated by heat.
Fremgangsmåten ifølge oppfinnelsen kan også benyttes til identifisering av numeriske kromosomale abnormaliteter, såsom Downs syndrom. I prenatal diagnostikk er det også mulig å bestemme hvorvidt fosteret er defekt, dvs. homozygot for et eller annet recessivt gen. The method according to the invention can also be used for the identification of numerical chromosomal abnormalities, such as Down's syndrome. In prenatal diagnostics, it is also possible to determine whether the fetus is defective, i.e. homozygous for some recessive gene.
Fremgangsmåten ifølge oppfinnelsen og nukleinsyrereagensene som anvendes i fremgangsmåten er beskrevet i nærmere detalj neden-under . The method according to the invention and the nucleic acid reagents used in the method are described in more detail below.
Eksempel 1 Example 1
Kvantifisering av en amplifisert onkogen Quantification of an amplified oncogene
a) Nukleinsyrereaqenser og deres fremstillinq a) Nucleic acid reagents and their preparation
STANDARDSONDER STANDARD PROBE
Celle- standardnukleinsyre Cell standard nucleic acid
c-Ki-rasI-genet er tilstede i alle menneskelige celler. Sondeparene for sandwich-hybridisering ble fremstilt ved subkloning av HindIII-fragmentet av c-Ki-rasI-genet som i lengde målte 3,8 kb, hvis restriksjonskart er blitt beskrevet av Chang et al., PNAS 79, pp 4848-52, 1982. Fragmentet er tilgjengelig f.eks. klonet inn i pBR322-plasmidet (ATCC 41032) og kan fås f.eks. fra The c-Ki-rasI gene is present in all human cells. The probe pairs for sandwich hybridization were prepared by subcloning the HindIII fragment of the c-Ki-rasI gene measuring 3.8 kb in length, the restriction map of which has been described by Chang et al., PNAS 79, pp 4848-52, 1982 The fragment is available e.g. cloned into the pBR322 plasmid (ATCC 41032) and can be obtained e.g. from
ATCC culture collection. ATCC culture collection.
Videre behandling av celle- standardnukleinsvren Further treatment of cell standard nucleic acid
pBR322-klonen beskrevet ovenfor ble behandlet med Bglll- og Hindlll-restriksjonsenzymer, og de resulterende fragmenter ble isolert fra agarosegel; rensede fragmenter som var anordnet nær hinannen ble subklonet inn i to egnede vektorer for fremstilling av detektor- og oppfangingssondene. The pBR322 clone described above was treated with BglII and HindIII restriction enzymes, and the resulting fragments were isolated from agarose gel; purified fragments that were arranged close to each other were subcloned into two suitable vectors for making the detector and capture probes.
Standard detektorsonde Standard detector probe
Et Bglll-Bglll-fragment som i lengde målte ca. 1,1 kb ble subklonet inn i BamHI-restriksjonsenzymsetet av pBR322-plasmidet og merket ved nick-translasjon med <125>I-merket dCTP. A Bglll-Bglll fragment measuring approx. 1.1 kb was subcloned into the BamHI restriction enzyme site of the pBR322 plasmid and labeled by nick translation with <125>I-labeled dCTP.
Standard oppfangingssonde Standard capture probe
Bglll-Hindlll-fragmentet på ca. 0,5 kb ble innsatt i M13 mplO-og mpll-fag-vektorer mellom restriksjonssetene for BamHI- og Hindlll-restriksjonsenzymene og festet til et nitrocellulosefilter (150 ng DNA/dia 1 cm). The Bglll-Hindlll fragment of approx. 0.5 kb was inserted into M13 mplO and mpll phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes and attached to a nitrocellulose filter (150 ng DNA/dia 1 cm).
FORSØKSSONDER EXPERIMENTAL PROBE
Forsøksnukleinsyre Experimental nucleic acid
Et sondepar for sandwich-hybridisering ble fremstilt fra et klonet c-myc-gen som kan fås f.eks. fra ATCC culture collection (ATCC 41010). Restriksjonskartet for genet er blitt beskrevet av Watt et al., PNAS 80, pp. 6307-6311, 1983. A probe pair for sandwich hybridization was prepared from a cloned c-myc gene which can be obtained e.g. from the ATCC culture collection (ATCC 41010). The restriction map for the gene has been described by Watt et al., PNAS 80, pp. 6307-6311, 1983.
Videre behandling av forsøksnukleinsyren Further treatment of the test nucleic acid
c-myc-genet ble behandlet med Hindlll-, Xbal- og Pstl-restriksjonsenzymer og fragmentene isolert fra agarosegelen, renset og subklonet inn i egnede vektorer for å fremstille detektor- og oppfangingssondene. The c-myc gene was treated with HindIII, XbaI and PstI restriction enzymes and the fragments isolated from the agarose gel, purified and subcloned into appropriate vectors to prepare the detector and capture probes.
Forsøks- detektorsonde Experimental detector probe
De enkelttrådede haler av Hindlll-Xbal-restriksjonsfragmentet av c-myc-genet, som målte 3,7 kb i lengde, ble gjort dobbelttrådede ved DNA-polymerase. Hindlll-linkerne ble innsatt ved T4-DNA-ligase i de resulterende DNA-fragmenter med butt ende; etter fenolekstraksjon ble DNA behandlet med Hindlll-restriksjonsenzymet. DNA-fragmentet ble deretter klonet inn i pBR322-plasmidet ved restriksjonssetet for Hindlll-restriksjonsenzymet og merket ved nick-translasjon med 125I-merket dCTP. The single-stranded tails of the HindIII-XbaI restriction fragment of the c-myc gene, measuring 3.7 kb in length, were made double-stranded by DNA polymerase. The HindIII linkers were inserted by T4 DNA ligase into the resulting blunt-ended DNA fragments; after phenol extraction, DNA was treated with the HindIII restriction enzyme. The DNA fragment was then cloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme and labeled by nick translation with 125 I-labeled dCTP.
Forsøks- oppfangingssonde Experimental capture probe
Det 1,1 kb store Xbal-Pstl-fragment av c-myc-genet ble klonet inn i M13 mplO- og mpll-fagkloningsvektorene mellom restriksjonssetene for Xbal- og Pstl-restriksjonsenzymene og festet til nitrocellulosefilteret (150 ng DNA/dia 1 cm). The 1.1 kb XbaI-Pstl fragment of the c-myc gene was cloned into the M13 mplO and mpll phage cloning vectors between the restriction sites of the XbaI and Pstl restriction enzymes and attached to the nitrocellulose filter (150 ng DNA/dia 1 cm) .
b) Bestemmelse av standardkurven b) Determination of the standard curve
Prøvene som ble benyttet til bestemmelse av standardkurven besto The samples used to determine the standard curve consisted of
av en alkalisk-denaturert pBR322-klon av c-myc-genet. Sandwich-hybridiseringsoppløsningen, til hvilken de ovenfor angitte forsøkssonder ble tilsatt, besto av 4 x SSC, 1 x Denhardts oppløsning, 200 /xg/ml sildesperma-DNA og 0,2 prosent SDS. Hybridisering fant sted ved 65°C i 17-19 timer, hvoretter filtrene ble vasket i vaskeoppløsningen (0,1 x SSC 0,2 prosent SDS) ved 50°C. Markøren som var festet til sandwichhybridene ble deretter tellet i gamma-telleren. of an alkaline-denatured pBR322 clone of the c-myc gene. The sandwich hybridization solution, to which the above test probes were added, consisted of 4 x SSC, 1 x Denhardt's solution, 200 µg/ml herring sperm DNA and 0.2 percent SDS. Hybridization took place at 65°C for 17-19 hours, after which the filters were washed in the washing solution (0.1 x SSC 0.2 percent SDS) at 50°C. The marker attached to the sandwich hybrids was then counted in the gamma counter.
c) Bestemmelse av antall gener c) Determination of the number of genes
Prøvene omfattet 1) celler fra en morkake fra menneske og 2) The samples included 1) cells from a human placenta and 2)
Colo 3 20-celler som kan fås f.eks. fra ATCC culture collection (ATCC-CCC220) . DNA ble isolert fra begge prøver, og den samme mengde celle-DNA denaturert ved alkalisk koking ble satt til i begge forsøk. Alkalisk denaturering hydrolyserte eventuelt RNA som forelå i prøven. Colo 3 20 cells which can be obtained e.g. from the ATCC culture collection (ATCC-CCC220). DNA was isolated from both samples, and the same amount of cell DNA denatured by alkaline boiling was added to both experiments. Alkaline denaturation hydrolyzed any RNA present in the sample.
Forsøket ble utført ved at der til hver prøve ble tilsatt både c-myc- og c-Ki-rasI-filtrene og de to merkede reagenser, hvilket tillot måling av både standard- og forsøks-DNA for hver prøve. På basis av c-Ki-rasI-bestemmelsene ble hver prøve funnet å inneholde den samme mengde DNA, og det kan derav sluttes at c-myc-genet i Colo 320-celler foreligger i et 16-20 ganger høyere kopitall enn i den normale situasjon. Resultatene er vist i tabell 2. The experiment was carried out by adding both the c-myc and c-Ki-rasI filters and the two labeled reagents to each sample, which allowed measurement of both standard and test DNA for each sample. On the basis of the c-Ki-rasI determinations, each sample was found to contain the same amount of DNA, and it can therefore be concluded that the c-myc gene in Colo 320 cells is present in a 16-20 times higher copy number than in the normal situation. The results are shown in table 2.
Eksempel 2 Example 2
Kvantifisering av amplifisert gen Quantification of amplified gene
a) Nukleinsyrereagenser og deres fremstilling a) Nucleic acid reagents and their preparation
STANDARDSONDER STANDARD PROBE
Celle- standardnukleinsvre Standard cell nucleic acid
Sammenligningsnukleinsyren ble tatt fra promotorområdet av metallotioningenet hos mus, dvs. MT-genet, og DNA umiddelbart på oversiden av det. Strukturen av MT-genet er blitt beskrevet av Pavlakis og Hamer, PNAS 80_, pp. 397-401, 1983. Referanse-nukleinsyrefragmentet er tilgjengelig f.eks. klonet inn i pBPV-MMTneo (342-12)-vektoren (ATCC 37224) og kan fås f.eks. fra ATCC culture collection. The reference nucleic acid was taken from the promoter region of the mouse metallothionein gene, i.e. the MT gene, and the DNA immediately upstream of it. The structure of the MT gene has been described by Pavlakis and Hamer, PNAS 80_, pp. 397-401, 1983. The reference nucleic acid fragment is available e.g. cloned into the pBPV-MMTneo (342-12) vector (ATCC 37224) and can be obtained e.g. from the ATCC culture collection.
Ytterligere behandling av celle- standardnukleinsyre Further processing of cell standard nucleic acid
Det MT-gen som er beskrevet ovenfor ble behandlet med Kpnl-, Bglll- og EcoRI-restriksjonsenzymer for subkloning inn i pAT153-plasmidet. Kpnl-halen ble omdannet til en Hindlll-hale med en linker. The MT gene described above was treated with KpnI, BglII and EcoRI restriction enzymes for subcloning into the pAT153 plasmid. The KpnI tail was converted to a HindIII tail with a linker.
Standard deteksionssonde Standard detection probe
EcoRI-Kpnl-(Hindlll)-fragmentet som målte ca. 1,2 kb og var anordnet på oversiden av promotorområdet for metallotioningenet ble klonet til pAT153-plasmidet mellom restriksjonssetene for EcoRI- og Hindlll-restriksjonsenzymene og merket ved nick-translasjon med <32>P-merket nukleosidtrifosfater. The EcoRI-KpnI-(HindIII) fragment measuring ca. 1.2 kb and was located upstream of the promoter region of the metallothionein gene was cloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindIII restriction enzymes and labeled by nick translation with <32>P-labeled nucleoside triphosphates.
Standard oppfangin<g>ssonde Standard capture probe
Kpnl-Bglll-fragmentet på 0,8 kb omfattende promotorområdet for metallotioningenet og området på oversiden av dette ble klonet inn i M13 mpl8- og M13 mpl9-fagvektorer mellom restriksjonssetene for Kpnl- og BamHI-restriksjonsenzymene og festet til nitrocellulosefilteret. The KpnI-Bglll fragment of 0.8 kb comprising the promoter region of the metallothionein gene and the region upstream thereof was cloned into M13 mpl8 and M13 mpl9 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes and attached to the nitrocellulose filter.
FORSØKSSONDER EXPERIMENTAL PROBE
Forsøksnukleinsvre Experimental nucleic acid
Sondeparet for sandwich-hybridiseringsforsøket ble fremstilt ved bruk av det i handelen tilgjengelige pMTVdhfr-plasmid (Betfiesda Research Laboratories, produkt nr. 53 69SS), hvilken struktur er beskrevet av Lee et al., Nature 294. pp. 228-232, 1981. The probe pair for the sandwich hybridization experiment was prepared using the commercially available pMTVdhfr plasmid (Betfiesda Research Laboratories, product no. 53 69SS), the structure of which is described by Lee et al., Nature 294. pp. 228-232, 1981.
Videre behandling av forsøksnukleinsyre Further processing of experimental nucleic acid
pMTVdhfr-plasmidet inneholdende cDNA av dihydrofolatreduktase-genet (DHFR) ble behandlet med Hindlll- og Bglll-restriksjonsenzymer. The pMTVdhfr plasmid containing the cDNA of the dihydrofolate reductase (DHFR) gene was treated with HindIII and BglII restriction enzymes.
Forsøks- deteksi onssonde Experimental detection probe
Hindlll-Bglll-fragmentet som målte 0,75 kb og svarte til området som koder for DHFR-genet av pMTVdhfr-plasmidet ble innsatt i plasmid pAT153-vektoren mellom restriksjonssetene for Hindlll-og BamHI-restriksjonsenzymene og merket ved nick-translasjon med <32>P-merket nukleosidtrifosfater. The HindIII-BglII fragment measuring 0.75 kb and corresponding to the region coding for the DHFR gene of the pMTVdhfr plasmid was inserted into the plasmid pAT153 vector between the restriction sites of the HindIII and BamHI restriction enzymes and tagged by nick translation with <32 >P-labeled nucleoside triphosphates.
Forsøks- oppfangingssonde Experimental capture probe
Et HindiII-fragment som målte 1,4 kb tatt fra MMTV-genområdet av pMTVdhfr-plasmidet ble klonet inn i M13 mpl8- og M13 mpl9-fagvektorene. A HindiII fragment measuring 1.4 kb taken from the MMTV gene region of the pMTVdhfr plasmid was cloned into the M13 mpl8 and M13 mpl9 phage vectors.
b) Bestemmelse av standardkurven b) Determination of the standard curve
Prøven som ble brukt i forsøket var renset DNA fra pMTVdhfr-plasmidet. Selve forsøket ble utført som beskrevet i eksempel lb, med den unntagelse at en væske-scintillasjonsteller ble brukt til telling. Den resulterende standardkurve er vist i tabell 3. The sample used in the experiment was purified DNA from the pMTVdhfr plasmid. The experiment itself was carried out as described in example 1b, with the exception that a liquid scintillation counter was used for counting. The resulting standard curve is shown in Table 3.
c) Bestemmelse av antallet gener c) Determination of the number of genes
Cellelinjer som var skaffet fra fibroblastcelle NIH 3T3 fra mus, Cell lines obtained from mouse fibroblast cell NIH 3T3,
hvilken cellelinje er tilgjengelig fra ATCC culture collection og som har nummer CRL 1658 og som var blitt transfisert med forskjellige mengder cDNA svarende til mRNA av DHFR-genet ble dyrket på celledyrkingsplater og benyttet som prøven. Cellene ble lysert ved bruk av natriumdodecylsulfat og deres DNA ble brutt opp ved anvendelse av skjærkrefter, idet de ble presset gjennom en fin hypoderm nål fra en injeksjonssprøyte. En 250 pl prøve svarende til ca. IO<6> celler ble tatt fra homogenatet og 50 /il NaOH tilsatt. Prøven ble kokt og nøytralisert med eddiksyre og hybridiseringsblandingen. Det samlede volum var 0,5 ml. Alle sondene som er beskrevet ovenfor ble tilsatt samtidig, og et såkalt blindfilter ble tilsatt som en bakgrunn-sammenlignings-prøve. Hybridisering, vasking og markørtelling ble utført som i which cell line is available from the ATCC culture collection and which has the number CRL 1658 and which had been transfected with different amounts of cDNA corresponding to mRNA of the DHFR gene was grown on cell culture plates and used as the sample. The cells were lysed using sodium dodecyl sulfate and their DNA was broken up using shear forces as they were pushed through a fine hypodermic needle from an injection syringe. A 250 pl sample corresponding to approx. 10<6> cells were taken from the homogenate and 50 µl NaOH added. The sample was boiled and neutralized with acetic acid and the hybridization mixture. The total volume was 0.5 ml. All the probes described above were added simultaneously, and a so-called blank filter was added as a background comparison sample. Hybridization, washing and marker counting were performed as in
eksempel lb, bortsett fra at en væske-scintillasjonsteller ble brukt til telling. Resultatene er vist i tabell 4. example lb, except that a liquid scintillation counter was used for counting. The results are shown in table 4.
MT-genet er en indre markør som måler antallet celler som foreligger i en prøve. Resultatene viser at i dette forsøk ga IO<6> celler et MT-spesifikt signal på 165 + 20 cpm. DHFR-reagensene måler mengden DHFR-cDNA. Antallet celler ble utledet fra det MT-spesifikke signal. Det var således mulig å bestemme antallet DHFR-cDNA-kopier i forskjellige cellelinjer som vist i tabell 4. The MT gene is an internal marker that measures the number of cells present in a sample. The results show that in this experiment IO<6> cells gave an MT-specific signal of 165 + 20 cpm. The DHFR reagents measure the amount of DHFR cDNA. The number of cells was deduced from the MT-specific signal. It was thus possible to determine the number of DHFR cDNA copies in different cell lines as shown in Table 4.
Eksempel 3 Example 3
Kvantifisering av budbringer RNA Quantification of messenger RNA
a) Nukleinsyrereagenser og deres fremstilling a) Nucleic acid reagents and their preparation
Ved bruk av forsøkssondene beskrevet i eksempel 2, er det også When using the experimental probes described in example 2, it is also
mulig å måle mengden mRNA som fås fra DHFR-cDNA. Strukturen av pMTVdhfr-plasmidet er slik at transkripsjonen av DHFR-genet begynner ved MMTV-promotoren. De resulterende budbringere er ca. 1,0 kb lange. Av dette fås 0,25 kb fra MMTV-promotorområdet og resten fra DHFR-cDNA (Lee et al., Nature 294, pp. 228-232, 1981). possible to measure the amount of mRNA obtained from DHFR cDNA. The structure of the pMTVdhfr plasmid is such that transcription of the DHFR gene begins at the MMTV promoter. The resulting messengers are approx. 1.0 kb long. Of this, 0.25 kb is obtained from the MMTV promoter region and the rest from the DHFR cDNA (Lee et al., Nature 294, pp. 228-232, 1981).
STANDARDSONDER STANDARD PROBE
Celle-standardnukleinsyren, standard-detektor- og standard-oppfangingssonden var som beskrevet i eksempel 2. The cell standard nucleic acid, standard detector and standard capture probe were as described in Example 2.
FORSØKSSONDER EXPERIMENTAL PROBE
Forsøksnukleinsyren, forsøks-detektor- og forsøks-oppfangings-sonden var som beskrevet i eksempel 2. The experimental nucleic acid, experimental detector and experimental capture probe were as described in Example 2.
b) Bestemmelse av standardkurven b) Determination of the standard curve
Prøven som ble brukt til standardkurve-bestemmelse besto av The sample used for standard curve determination consisted of
budbringer RNA svarende til dihydrofolatreduktase-genet fremstilt ved in vitro transkripsjon. Det DNA som var nødvendig for transkripsjon ble fremstilt ved subkloning av 1,4 kb Hindlll-fragmentet av MMTV-promotoren av pMTVdhfr-plasmidet og 0,75 kb Hindlll-Bglll-fragmentet (DHFR-cDNA) ved siden av hverandre inn i pSP64-plasmidet (Promega Biotec) mellom restriksjonssetene for Hindlll- og BamHI-restriksjonsenzymene. Prøve-RNA ble lagret i 0,2 prosent SDS vandig oppløsning. messenger RNA corresponding to the dihydrofolate reductase gene produced by in vitro transcription. The DNA required for transcription was prepared by subcloning the 1.4 kb HindIII fragment of the MMTV promoter of the pMTVdhfr plasmid and the 0.75 kb HindIII-BglII fragment (DHFR cDNA) side by side into pSP64- plasmid (Promega Biotec) between the restriction sites of the HindIII and BamHI restriction enzymes. Sample RNA was stored in 0.2 percent SDS aqueous solution.
Sandwich-hybridiseringsforsøket ble utført som beskrevet i eksempel lb og 2b, men denatureringen ble sløyfet. The sandwich hybridization experiment was performed as described in examples 1b and 2b, but the denaturation was omitted.
c) Bestemmelse av antallet budbringer RNA- molekvler Antallet budbringer RNA-molekyler som svarer til DHFR-genet, ble c) Determination of the number of messenger RNA molecules The number of messenger RNA molecules corresponding to the DHFR gene was
bestemt fra cellelinjene som beskrevet i eksempel 2. determined from the cell lines as described in Example 2.
Cellene ble lysert ved bruk av natriumdodecylsulfat, og deres DNA ble underkastet lett skjærbehandling ved pressing gjennom en fin hypoderm nål fra en injeksjossprøyte. En 250 /xl prøve av homogenatet ble tatt, svarende til ca. 5 x IO<6> celler. Homogenatet ble deretter satt til sandwich-hybridiseringsforsøket uten denaturering. Sandwich-hybridisering fant sted som beskrevet i eksempel 2c og lb, med den unntagelse at bare DHFR-sondene ble satt til hybridiseringsoppløsningen. I en parallell prøve på 250 jul homogenat ble celleantallet bestemt ved bruk av MT-sonden som beskrevet i eksempel 2c. The cells were lysed using sodium dodecyl sulfate, and their DNA was subjected to light shearing by pressing through a fine hypodermic needle from a hypodermic syringe. A 250 µl sample of the homogenate was taken, corresponding to approx. 5 x IO<6> cells. The homogenate was then added to the sandwich hybridization experiment without denaturation. Sandwich hybridization took place as described in Examples 2c and 1b, with the exception that only the DHFR probes were added to the hybridization solution. In a parallel sample of 250 µl homogenate, the cell count was determined using the MT probe as described in example 2c.
Resultatene er vist i tabell 6. The results are shown in table 6.
Resultatene viste at cellelinje I produserte pr. celle ca. 100 budbringer RNA-molekyler fra DHFR-genene, og cellelinje II produserte ca. 500 budbringer RNA-molekyler fra DHFR-genene. The results showed that cell line I produced per cell approx. 100 messenger RNA molecules from the DHFR genes, and cell line II produced approx. 500 messenger RNA molecules from the DHFR genes.
Eksempel 4 Example 4
Kvantifisering av amplifisert gen ved oppløsnings-hybridisering Quantification of amplified gene by resolution hybridization
a) Nukleinsvrereagenser og deres fremstilling a) Nucleic acid reagents and their preparation
STANDARDSONDER STANDARD PROBE
Celle-standardnukleinsyre, standard-detektor- og standard-oppfangingssonde var som beskrevet i eksempel 2. 1,2 kb EcoRI-Kpnl-(Hindlll)-fragmentet i pAT153 ble merket ved nick-translasjon med <125>I-merket deoksycytidin. De 0,8 kb Kpnl-Bglll-fragmenter i M13 mpl8 og M13 mpl9 ble modifisert med biotin ved bruk av Photoprobe™-reagensen (Vector Laboratories, CA, USA, produktnr. SP-1000). Cell standard nucleic acid, standard detector and standard capture probe were as described in Example 2. The 1.2 kb EcoRI-Kpnl-(HindIII) fragment in pAT153 was labeled by nick translation with <125>I-labeled deoxycytidine. The 0.8 kb Kpnl-Bglll fragments in M13 mpl8 and M13 mpl9 were modified with biotin using the Photoprobe™ reagent (Vector Laboratories, CA, USA, Product No. SP-1000).
FORSØKSSONDER EXPERIMENTAL PROBE
Forsøksnukleinsyren, forsøks-detektor- og forsøks-oppfangings-sonden var som beskrevet i eksempel 2. 0,75 kb Hindlll-Bglll-fragmentet i pAT153 ble merket med <125>I-merket deoksycytidin. 1,4 kb HindiII-fragmentene i M13 mpl8 og M13 mpl9 ble biotiny-lert ved bruk av Photoprobe™ som angitt ovenfor. The test nucleic acid, test detector and test capture probe were as described in Example 2. The 0.75 kb HindIII-BglII fragment in pAT153 was labeled with <125>I-labeled deoxycytidine. The 1.4 kb HindiII fragments in M13 mpl8 and M13 mpl9 were biotinylated using Photoprobe™ as indicated above.
b) Bestemmelse av standardkurvene b) Determination of the standard curves
En celle-standardkurve ble fremstilt ved bruk av en kjent mengde A cell standard curve was prepared using a known quantity
celler, fra hvilke hybridiseringssignalet ble målt ved bruk av standardsondene som kjente igjen MT-genet. En forsøksnuklein-syre-standardkurve ble fremstilt med pMTVdhfr-plasmidet og forsøkssondene som kjente igjen dette plasmid. Hybridiseringer ble utført i 200 pl av en oppløsning bestående av 0,6 M NaCl, 20 mM fosfatbuffer, pH 7,5, 1 mM EDTA, 4 prosent polyetenglykol (PEG 6000) i 1,5 timer ved 70°C. Etter reaksjonen ble 50 jul streptavidin-agarose (Bethesda Research Laboratories, Maryland, USA, produktnr. 5942SA), og 1 M NaCl, 10 mM natriumfosfat, pH 7,5, 1 mM EDTA satt til det endelige volum på 500 ul. Hybridene ble samlet opp på streptavidin-agarosen ved 37°C i 15 minutter. Agarosen ble vasket én gang i 5 minutter med den buffrede 1 M NaCl-oppløsning ved 37°C og to ganger i 2 minutter med 15 mM NaCl, 1,5 mM natriumcitrat ved 55°C. Mengden bundne hybrider ble bestemt ved måling av agarosen i en gammateller. (Syvanenen et al., Nucleic Acids Res. 14, 5037-5048, 1986). Resultatene er vist i tabell 7 og 8. cells, from which the hybridization signal was measured using the standard probes that recognized the MT gene. An experimental nucleic acid standard curve was prepared with the pMTVdhfr plasmid and the experimental probes that recognized this plasmid. Hybridizations were performed in 200 µl of a solution consisting of 0.6 M NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4 percent polyethylene glycol (PEG 6000) for 1.5 hours at 70°C. After the reaction, 50 µl of streptavidin-agarose (Bethesda Research Laboratories, Maryland, USA, product no. 5942SA), and 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA were added to the final volume of 500 µl. The hybrids were collected on the streptavidin-agarose at 37°C for 15 minutes. The agarose was washed once for 5 min with the buffered 1 M NaCl solution at 37°C and twice for 2 min with 15 mM NaCl, 1.5 mM sodium citrate at 55°C. The amount of bound hybrids was determined by measuring the agarose in a gamma counter. (Syvanenen et al., Nucleic Acids Res. 14, 5037-5048, 1986). The results are shown in tables 7 and 8.
c) Bestemmelse av antallet gener c) Determination of the number of genes
Prøver av cellelinjene beskrevet i eksempel 2 ble behandlet på Samples of the cell lines described in Example 2 were treated on
lignende måte, bortsett fra at volumet pr. prøve svarende til ca. 2 x IO<6> celler var 125 fil. Bestemmelsen av antall celler og antall forsøksnukleinsyre-molekyler ble utført i separate ampuller ved tilsetning av celleprøven, de riktige detektor- og oppfangingssonder og komponentene av hybridiseringsblandingen til et endelig volum på 200 fil. Sammenligningsprøver uten cellestandard eller forsøks-DNA ble inkludert. Hybridisering, oppsamling av hybrider, vasking og måling ble utført som beskrevet i eksempel 4b. Resultatene ble avlest fra standardkurver fremstilt i parallell som beskrevet i eksempel 4b. Resultatene er vist i tabell 9. similar way, except that the volume per sample corresponding to approx. 2 x IO<6> cells were 125 fil. The determination of the number of cells and the number of test nucleic acid molecules was carried out in separate vials by adding the cell sample, the appropriate detector and capture probes and the components of the hybridization mixture to a final volume of 200 μl. Control samples without cell standard or experimental DNA were included. Hybridization, collection of hybrids, washing and measurement were performed as described in Example 4b. The results were read from standard curves prepared in parallel as described in example 4b. The results are shown in table 9.
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US5232829A (en) * | 1989-09-29 | 1993-08-03 | Hoffmann-La Roche Inc. | Detection of chlamydia trachomatis by polymerase chain reaction using biotin labelled lina primers and capture probes |
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US5580971A (en) * | 1992-07-28 | 1996-12-03 | Hitachi Chemical Company, Ltd. | Fungal detection system based on rRNA probes |
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