SE463212B - METHOD FOR IDENTIFYING NUCLEIC ACIDS - Google Patents
METHOD FOR IDENTIFYING NUCLEIC ACIDSInfo
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- SE463212B SE463212B SE8600011A SE8600011A SE463212B SE 463212 B SE463212 B SE 463212B SE 8600011 A SE8600011 A SE 8600011A SE 8600011 A SE8600011 A SE 8600011A SE 463212 B SE463212 B SE 463212B
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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
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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
- C12Q1/6813—Hybridisation assays
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Description
463 212 2 kommes inte en tillräcklig hybridisering på mindre än 12 timmar. När tvåstegs-sandwich-hybridisering (Dunn & Hassell, Cell. volym 12, p. 23-36, l977) genomföres erfordras en hybridiseringstid av minst 24 timmar. 463 212 2 sufficient hybridization of less than 12 is not obtained hours. When two-step sandwich hybridization (Dunn & Hassell, Cell. volume 12, pp. 23-36, 1977) is required hybridization time of at least 24 hours.
Minst tvâ sonder användes företrädesvis vid metoden enligt uppfinningen. Sonderna är nukleinsyrafragment tillräckligt homologa till målnukleinsyran. Det är fördelaktigt, men inte nödvändigt, att sonderna är homologa till bindningsställena, vilka är belägna relativt nära varandra i nukleinsyran som skall identifieras. Sonderna behöver inte överlappa varandra.At least two probes are preferably used in the method of the invention. The probes are nucleic acid fragments sufficient homologous to the target nucleic acid. It is beneficial, but not necessary that the probes are homologous to the binding sites, which are located relatively close to each other in the nucleic acid as shall be identified. The probes do not have to overlap.
Sonderna kan framställas syntetiskt eller semi-syntetiskt genom rekombinant-DNA-tekniker eller från nukleinsyror isole- rade direkt från naturen. Sonderna är även kommersiellt till- gängliga från flera källor. En sond kan vara bunden till en lämplig vektor. Den kan innehålla vektordelar eller vara fullständigt utan vektordelar.The probes can be made synthetically or semi-synthetically by recombinant DNA techniques or from nucleic acids isolated directly from nature. The probes are also commercially available. available from several sources. A probe can be tied to one appropriate vector. It can contain vector parts or be completely without vector parts.
Detektorsonden är märkt med en lämplig markör. Olika radio- aktiva isotoper eller radioaktivt märkta föreningar kan användas som markörer. Markörsubstansen kan även vara fluorescerande, luminiscerande, ljusemitterande, enzymatiskt eller immunologiskt påvisbar, etc. Markörer baserade på biotin och advidin eller streptavidin, lantanidkelater, ferritin och hemeföreningar och immunologiskt pâvisbara haptener såsom acetoxiacetylfluorenderivat (WO 8302286) kan omnämnas som exempel. Identifiering genom mediering av proteiner är även möjlig. Sättet enligt uppfinningen beror inte på den använda markören. Alla hittills kända markörsubstanser lämpliga för nukleinsyrahybridisering eller de som utvecklas i framtiden kan fritt appliceras till metoden.The detector probe is marked with a suitable marker. Various radio active isotopes or radiolabeled compounds can be used as markers. The marker substance can also be fluorescent, luminescent, light emitting, enzymatic or immunologically detectable, etc. Markers based on biotin and advidin or streptavidin, lanthanide chelates, ferritin and home compounds and immunologically detectable haptens such as acetoxyacetylfluorene derivatives (WO 8302286) may be mentioned as example. Identification by mediation of proteins is also Possible. The method according to the invention does not depend on the one used the cursor. All hitherto known marker substances suitable for nucleic acid hybridization or those that will be developed in the future can be freely applied to the method.
Till den andra sonden, den s.k. infångningssonden är en kom- ponent med affinitet till en annan komponent bunden. Biotin - avidin eller streptavidin, tungmetallderivat - tiogrupper, olika homopolynukleotider, såsom poly dG - poly dC, poly dA - poly dT och poly dA - poly U är lämpliga affinitetspar. Men* _ 3 463 212 även andra affinitetspar kan användas, förutsatt att kompo- nenterna har tillräckligt stark affinitet till varandra.To the second probe, the so-called the capture probe is a component with affinity bound to another component. Biotin - avidin or streptavidin, heavy metal derivatives - thio groups, different homopolynucleotides, such as poly dG - poly dC, poly dA - poly dT and poly dA - poly U are suitable affinity pairs. Men * _ 3 463 212 other affinity pairs can also be used, provided that the have a sufficiently strong affinity for each other.
Lämpliga affinitetspar finns bland ligander och konjugat som användes i de immunologiska metoderna.Suitable affinity pairs are found among ligands and conjugates such as used in the immunological methods.
Innan hybridiseringsreaktionen sker behandlas provet för att frigöra målnukelinsyrorna i enkelsträngad form i hybridise- ringslösningen. Hybridiseringen sker i en hybridiserings- blandning i vilken målnukelinsyrorna,_den märkta sonden och infångningssonden, när så erfordras, gjorts enkelstränga- de. Olika lämpliga buffertmedel kan användas som hybridise- ringslösningar. Hybridiseringen sker inom temperaturområdet O-80°C, men det är fördelaktigt att använda en temperatur av 65°C. Om hybridiseringslösningen innehåller formamid (40-55 %) kan en temperatur av 37°C användas. En timme är tillräcklig som hybridiseringstid.Before the hybridization reaction takes place, the sample is treated to release the target nucleic acids in single-stranded form in hybridized the ring solution. The hybridization takes place in a hybridization mixture in which the target nucleic acids, the labeled probe and the capture probe, when required, is made single-stranded the. Various suitable buffering agents can be used as hybridizers. solutions. The hybridization takes place within the temperature range O-80 ° C, but it is advantageous to use a temperature of 65 ° C. If the hybridization solution contains formamide (40-55%) a temperature of 37 ° C can be used. One hour is enough as hybridization time.
När hybridiseringen sker spädes lösningen när så erfordras för att göra betingelserna fördelaktiga för affinitetsparet.When the hybridization takes place, the solution is diluted when required to make the conditions favorable for the affinity pair.
Därefter bringas blandningen i kontakt med den andra medlemmen av affinitetsparet. Affinitetskromatografikolonner, filter, plastytor, glasytor, etc. kan användas för att uppfånga upp- fångningssonden:målnukleinsyranzdetektorsondhybriden.The mixture is then brought into contact with the other member of the affinity pair. Affinity chromatography columns, filters, plastic surfaces, glass surfaces, etc. can be used to intercept the capture probe: the target nucleic acid detector probe hybrid.
Bärarmaterialet i affinitetskolonnen kan exempelvis vara cellulosa, latex, polyakrylamid, dextran eller agaros. Dessa material kan även användas som suspension i ett teströr. Det är även fördelaktigt att använda teströr med den andra kompo- nenten av affinitetsparet fixerad till dess inre yta. Det är en nödvändig förutsättning för det valda materialet att det är möjligt att fixera en komponent med affinitet för komponenten bunden till infångningssonden till detta.The carrier material in the affinity column may be, for example cellulose, latex, polyacrylamide, dextran or agarose. These material can also be used as a suspension in a test tube. The is also advantageous to use test tubes with the other component. of the affinity pair fixed to its inner surface. It is a necessary condition for the selected material that it is possible to fix a component with affinity for the component bound to the capture probe for this.
Om provet innehåller nukleinsyran som skall identifieras erhålles en infångningssond:målnukleinsyra:detektorsond-hybrid från hybridiseringen. Under fraktionering vidhäftar denna hyb- rid till bäraren. Den markör av fraktionen som vidhäftar till bäraren kan mätas enligt konventionella metoder direkt från 463 212 bäraren eller efter eluering från den eluerade lösningen.If the sample contains the nucleic acid to be identified a capture probe: target nucleic acid: detector probe hybrid is obtained from hybridization. During fractionation, this hybrid adheres ride to the carrier. The marker of the fraction that adheres to the carrier can be measured according to conventional methods directly from 463 212 the vehicle or after elution from the eluted solution.
Andra system, exempelvis fasextraktion eller magnetiska fält kan även användas istället för affinitetskromatografi i frak- tioneringen.Other systems, such as phase extraction or magnetic fields can also be used instead of affinity chromatography in fractions. tionation.
Sättet enligt uppfinningen beskrives mera detaljerat i följan- de exempel. Sättet enligt uppfinningen är inte beroende av nuklein- syrafragmenten som användes i exemplen.The method according to the invention is described in more detail in the following. the examples. The method according to the invention does not depend on the acid fragments used in the examples.
Exempel l Identifiering av adenovirus-DNA från ett cellysat genom syran av homopolynukleotider Den använda detektorsonden är 125 I-märkt rekombinantfag mKTH 1206, som innehåller ett Bgl II-fragment av adenovirusgenomet från position 42 - 45,3 % på genmönstret av adenovirus typ 2.Example 1 Identification of adenovirus DNA from a cell lysate by the acid of homopolynucleotides The detector probe used is 125 I-labeled recombinant phage mKTH 1206, which contains a Bgl II fragment of the adenovirus genome from position 42 - 45.3% on the gene pattern of adenovirus type 2.
Rekombinantfagen har deponerats vid depositionsinstitutionen Deutsche Sammlung von Mikroorganismen under nummer DSM 2827.The recombinant phage has been deposited at the depository institution Deutsche Sammlung von Mikroorganismen under number DSM 2827.
Dess specifika aktivitet är 7 x 107 cpm/pg DNA. Sonden beskri- ves mera detaljerat i Ranki et al Gene 21, p. 77-85, 1983.Its specific activity is 7 x 107 cpm / pg DNA. Probe described see in more detail in Ranki et al Gene 21, pp. 77-85, 1983.
Den använda infångningssonden är rekombinantplasmiden pKTH 1202, som deponerats vid depositionsinstitutionen Deutsche Samm- lung von Mikroorganismen under nummer DSM 2824 och innefattar ett BamHI D-fragment av adenoviruset (mönsterposition 29 - 42 ) klonad i plasmiden pBR322. Rekombinantplasmiden pKTH 1202 (DSM 2824) fragmenterades med användning av restriktions- % enzymet Hae III och en poly A-svans bands till 3'-ändarna av fragmenten med användning av terminaltransferasenzym. Svansens längd mättes genom 3H-A-inkorporation. Längden var i medeltal ungefär 70 A-rester. Innan den användes denaturerades infång- ningssonden genom kokning.The capture probe used is the recombinant plasmid pKTH 1202. deposited with the depositary Deutsche Samm- lung von Microorganism under number DSM 2824 and includes a BamHI D fragment of the adenovirus (pattern position 29 - 42 ) cloned into the plasmid pBR322. Recombinant plasmid pKTH 1202 (DSM 2824) were fragmented using restriction % the enzyme Hae III and a poly A tail were bound to the 3 'ends of the fragments using terminal transferase enzyme. The tail length was measured by 3 H-A incorporation. The length was average about 70 A residues. Before use, the captured by boiling.
Det använda provet bestod av adenovirus-infekterade A-549- celler. Cellerna inkuberades i 21 timmar efter infektionen.The sample used consisted of adenovirus-infected A-549 cells. The cells were incubated for 21 hours after infection.
Cellerna samlades och lyserades med användning av en 1%-íg natriumdodecylsulfatlösning. Lösningen innehöll ungefär 106 celler/ml. Dess viskositet sänktes genom sonikering. Iden- 465 212 tiskt behandlade icke-infekterade A-549-celler användes som kontroll. Innan hybridisering kokades provet i 5 minuter i 0,02M NaOH, kyldes till 0°C och neutraliserades med ättik- syra.The cells were collected and lysed using a 1% Ig sodium dodecyl sulfate solution. The solution contained about 106 cells / ml. Its viscosity was lowered by sonication. Iden- 465 212 treated non-infected A-549 cells were used as Control. Before hybridization, the sample was boiled for 5 minutes in 0.02M NaOH, cooled to 0 ° C and neutralized with acetic acid. acid.
För testet kombinerades 500.000 cpm detektorsond, 50 ng in- fångningssond DNA och 10 pl prov i ett teströr. volymen justerades till 50 ul och den använda buffrade lösningen var 0,6M natriumklorid, 0,06M natriumcitrat, 0,02M natrium, fos- fat (pH 7,6) och 0,5 % natriumdodecylsulfat. Blandningen in- kuberades i en timme vid 65°C.For the test, a 500,000 cpm detector probe, 50 ng in- capture probe DNA and 10 μl sample in a test tube. volumes was adjusted to 50 μl and the buffered solution used was 0.6M sodium chloride, 0.06M sodium citrate, 0.02M sodium, phosphorus barrels (pH 7.6) and 0.5% sodium dodecyl sulfate. The mixture is was incubated for one hour at 65 ° C.
Efter hybridisering kyldes lösningen till +20°C och fick sakta rinna genom en kromatografikolonn med l ml oligo dT-cellulosa.After hybridization, the solution was cooled to + 20 ° C and allowed to cool slowly flow through a chromatography column with 1 ml of oligo dT-cellulose.
Lösningen som fick passera genom återvanns och kördes ännu en gång genom kolonnen. Därefter tvättades kolonnen med 20 ml av en lösning innehållande 0,l5M natriumklorid, 0,0l5M natrium- fosfat (pH 7,6) och 0,5 % natriumdodecylsulfat. Testad DNA frigjordes slutligen med användning av l ml 0,02M NaOH. Denna lösning återvanns och dess radioaktivitet bestämdes. 125 Resultat: I-aktivitet (cpm) 83-89, 1984.The solution that had to pass through was recovered and run another walk through the column. Then the column was washed with 20 ml of a solution containing 0.5M M sodium chloride, 0.05M M sodium chloride phosphate (pH 7.6) and 0.5% sodium dodecyl sulphate. DNA tested was finally released using 1 ml of 0.02M NaOH. This solution was recovered and its radioactivity was determined. 125 Result: I activity (cpm) 83-89, 1984.
Målnukelinsyra:infekterade celler Kontrollceller 5230 325 Exempel 2 Identifiering av DNA av Chlamydia trachomatis med hjälp av biotin-streptavidin Den använda detektorsonden var 1251-märkt rekombinantfag mKTH 1245, innehållande två BamHI - SalI DNA-fragment från klonen pKTH 1220, vilka tillsammans är bundna till Ml3mp8-vektorn.Target nucleic acid: infected cells Control cells 5230 325 Example 2 Identification of DNA of Chlamydia trachomatis using biotin-streptavidin The detector probe used was 1251-labeled recombinant phage mKTH 1245, containing two BamHI - SalI DNA fragments from the clone pKTH 1220, which are together bound to the M13mp8 vector.
Klonen pKTH 1220 har deponerats vid depositionsinstitutionen Deutsche Sammlung von Mikroorganismen under nummer DSM 2825 och beskrives i Palva et al FEMS Microbiology Letters 22, p. 463 212 6 Den använda infångningssonden var rekombinantplasmiden pKTH 1250. Denna plasmid består av 2,9 kb SalI - ClaI-fragment från plasmiden pKTH 1220 (DSM 2825) och av vektorn pAT 153. Biotin- molekyler bands kovalent till pKTH 1250 DNA med användning av den kända"nickïtranslationsmetoden (Rigby et al., J. Mol.The clone pKTH 1220 has been deposited at the depository institution Deutsche Sammlung von Mikroorganismen under number DSM 2825 and described in Palva et al FEMS Microbiology Letters 22, p. 463 212 6 The capture probe used was the recombinant plasmid pKTH 1250. This plasmid consists of 2.9 kb SalI - ClaI fragment from plasmid pKTH 1220 (DSM 2825) and of the vector pAT 153. Biotin molecules were covalently linked to pKTH 1250 DNA using the known "nickit translation method" (Rigby et al., J. Mol.
Biol. 113, p. 237-251, 1977) och biotin-ll-UTP som substrat (Bethesda Research Laboratories). Infångningssond-DNA kokades i en buffert innefattande 10 mM tris-Cl, pH 7,6, 1 mM EDTA, 5 minuter innan användningen.Biol. 113, pp. 237-251, 1977) and biotin-II-UTP as substrate (Bethesda Research Laboratories). Capture probe DNA was boiled in a buffer comprising 10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 5 minutes before use.
Målnukleinsyran var rekombinantplasmiden pKTH 1220 (DSM 2825).The target nucleic acid was the recombinant plasmid pKTH 1220 (DSM 2825).
Denna plasmid innehåller ungefär 10 kb av DNA-karaktäristiken av Chlamydia trachomatis, bunden till vektorn pBR322. Plasmi- den tjänar som en modell-DNA, representerande bakteriens genom-DNA. Innan användningen kokades plasmiden i 5 minuter i 0,02M NaOH varefter lösningen neutraliserades med ättiksyra.This plasmid contains approximately 10 kb of the DNA characteristic of Chlamydia trachomatis, bound to the vector pBR322. Plasma it serves as a model DNA, representing the bacterium genomic DNA. Before use, the plasmid was boiled for 5 minutes in 0.02M NaOH after which the solution was neutralized with acetic acid.
Streptavidin, som användes som affinitetsmateríal, fixerades till CNBr-aktiverad Sepharose (Pharmacia) enligt Axen et al., Nature 214, p. 1302-1304, 1967.Streptavidin, which was used as an affinity material, was fixed to CNBr-activated Sepharose (Pharmacia) according to Axen et al., Nature 214, pp. 1302-1304, 1967.
För testet kombinerades 500.000 cpm sond, 50 ng infångnings- sond-DNA och 10 ng mål-DNA i ett teströr. Volymen justerades till 20 ul och buffertlösningen var samma som i exempel l.For the test, a 500,000 cpm probe, 50 ng of capture probe DNA and 10 ng of target DNA in a test tube. The volume was adjusted to 20 μl and the buffer solution was the same as in Example 1.
Kontroll-DNA var kalvtymus-DNA.Control DNA was calf thymus DNA.
Blandningen inkuberades i 60 minuter vid 65°C. Därefter tillsattes 500 ul av en buffertlösning med sammansättningen 0,lM tris-Cl, pH 7,5, 0,1M NaCl, 2mM MgC12, 0,05 % Triton x-100. Slutligen fraktionerades lösningen i en 0,2 ml strep- tavidin-Sepharose-kolonn. Kolonnen tvättades med 10 ml av ovannämnda buffert och 10 ml 0,0l5M natriumklorid, 0,015M natriumfosfat (pH 7,6), 0,5 % natriumdodecylsulfat (50°C).The mixture was incubated for 60 minutes at 65 ° C. Then 500 μl of a buffer solution with the composition was added 0.1M Tris-Cl, pH 7.5, 0.1M NaCl, 2mM MgCl 2, 0.05% Triton x-100. Finally, the solution was fractionated in a 0.2 ml tavidine-Sepharose column. The column was washed with 10 ml of the above buffer and 10 ml of 0.05M sodium chloride, 0.015M sodium phosphate (pH 7.6), 0.5% sodium dodecyl sulfate (50 ° C).
Således vidhäftade det biotinylerade DNA till streptavidinen under det att det andra DNA passerade genom kolonnen. Den radioaktiva sonden vidhäftade endast som ett resultat av I hybridbildning. Den infångade radioaktiviteten bestämdes genom§ | ___» att hela kolonnen överfördes i ett räknerör av en gamma- 7 463 212 räknare. l25I-aktivitet (cpm) Resultat: Målnukleinsyra: ng pKTH 1220 10 ng kontroll-DNA 1350 115 Exempel 3 Identifiering av plasmiden pBR322 DNA med hjälp av ett anti- gen-antikropp-par Den använda detektorsonden är ett derivat av plasmiden pBR322 (kommersiellt tillgänglig från flera källor) från vilken PstI - Sa1I (3613 - 615) fragment avlägsnats. Plasmiden märktes med fotobiotin med användning av en känd metod (Forster et al., Nucleic Acids Res. 13, p. 745-761, 1985) och kommersiellt reagens (Bresa, Adelaide, Australien).Thus, the biotinylated DNA adhered to the streptavidin while the other DNA passed through the column. The the radioactive probe adhered only as a result of I hybrid formation. The captured radioactivity was determined by§ | ___ » that the whole column was transferred in a counter tube of a gamma- 7 463 212 counter. 125 I activity (cpm) Result: Target nucleic acid: ng pKTH 1220 10 ng control DNA 1350 115 Example 3 Identification of the plasmid pBR322 DNA by means of an antibody gene-antibody pair The detector probe used is a derivative of the plasmid pBR322 (commercially available from several sources) from which PstI - Sa1I (3613 - 615) fragments removed. The plasmid was labeled photobiotin using a known method (Forster et al., Nucleic Acids Res. 13, pp. 745-761, 1985) and commercially reagent (Bresa, Adelaide, Australia).
Den använda infångningssonden var DNA från en rekombinantfag Ml3mp11 i vilken pBR322 PstI - SalI-fragment introducerats.The capture probe used was DNA from a recombinant phage M13mp11 in which pBR322 PstI - SalI fragment was introduced.
DNA:et hade sulfonerats med användning av en känd metod (Orgenics Ltd., Yavne, Israel).The DNA had been sulfonated using a known method (Orgenics Ltd., Yavne, Israel).
Provet var E. coli HBl0l, som bar plasmiden pBR322, vars mängd ökats med användning av kloramfenikolamplifiering (Maniatis et al., Molecular cloning, A laboratory manual, Cold Spring Har- bor Laboratory 1982). Bakteriecellerna lyserades med lysozym följt av kokning i NaOH som beskrivits i publikationen Palva, J. Clin. Microbiol. 18, p. 92-100, 1983.The sample was E. coli HB101, which carried the plasmid pBR322, the amount of which increased using chloramphenicol amplification (Maniatis et al., Molecular cloning, A laboratory manual, Cold Spring Har- bor Laboratory 1982). The bacterial cells were lysed with lysozyme followed by boiling in NaOH as described in the publication Palva, J. Clin. Microbiol. 18, pp. 92-100, 1983.
Antisulfonmonoklonala antikroppar användes för att belägga polystyrenmikrotiterbrunnar enligt standardmetoder (McKearn, ii Hybridomas: A New Dimension in Biological Analyses, ed. ¿ Kennett et al., Plenum Press 1980). ; F För testet kombinerades 5 x 106 lyserade E. coli-celler (både É med och utan pBR322) med 100 ng vardera av infångnings-DNA och: _ nu ___4 465 212 8 detektorsond i en 50 pl hybridiseringsblandning. Betingelserna var som i exempel l förutom att 5 % polyetylenglykol (PEG 6000) sattes till blandningen och natriumdodecylsulfatkoncen- trationen var 0,1 %. Efter hybridisering späddes lösningen till 250 ul genom tillsats av 0,02M natriumfosfat (pH 7,6), varefter lösningen överfördes till antikroppbelagda mikro- titerbrunnar. Detta följdes av inkubation i 2 timmar vid 37°C.Antisulfone monoclonal antibodies were used to coat polystyrene microtiter wells according to standard methods (McKearn, ii Hybridomas: A New Dimension in Biological Analyzes, ed. ¿ Kennett et al., Plenum Press 1980). ; F For the test, 5 x 10 6 lysed E. coli cells (both É with and without pBR322) with 100 ng each of capture DNA and: _ nu ___4 465 212 8 detector probe in a 50 μl hybridization mixture. The conditions was as in Example 1 except that 5% polyethylene glycol (PEG 6000) was added to the mixture and the sodium dodecyl sulfate concentration the tration was 0.1%. After hybridization, the solution was diluted to 250 μl by adding 0.02M sodium phosphate (pH 7.6), after which the solution was transferred to antibody-coated micro- titer wells. This was followed by incubation for 2 hours at 37 ° C.
Brunnen tvättades därefter väl med en lösning innehållande 0,l5M natriumklorid, 0,02M natriumfosfat, pH 7,6 och 0,05% triton X-100. Närvaron av detektorsonden visualiserades genom tillsats av streptavidin (Bethesda Research Laboratories BRL), tvättning, tillsats av biotinylerad alkalifosfatas (BRL) och tvättning såsom beskrivits av Leary et al., Proc. Natl. Acad.The well was then washed well with a solution containing 0.5M sodium chloride, 0.02M sodium phosphate, pH 7.6 and 0.05% triton X-100. The presence of the detector probe was visualized by addition of streptavidin (Bethesda Research Laboratories BRL), washing, addition of biotinylated alkali phosphatase (BRL) and washing as described by Leary et al., Proc. Natl. Acad.
Sci. USA 80, p. 4045-4049,l983. Slutligen tillsattes 250 pl av en 35 mg/ml paranitrofenylfosfat (Sigma)-lösning i dietanol- aminbuffert (pH 10). Efter 60 minuter upphörde reaktionen och absorbansen mättes vid 410 nm.Sci. USA 80, pp. 4045-4049, l983. Finally, 250 μl of was added a 35 mg / ml paranitrophenyl phosphate (Sigma) solution in diethanol- amine buffer (pH 10). After 60 minutes the reaction stopped and the absorbance was measured at 410 nm.
BÉÉEÄÉÉÉ= A410 nm Målnukleinsyra: celler med pBR322 Celler utan pBR322 )2 0,15BÉÉEÄÉÉÉÉ = A410 nm Target nucleic acid: cells with pBR322 Cells without pBR322 ) 2 0.15
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI850004A FI72146C (en) | 1985-01-02 | 1985-01-02 | Procedure for Identifying Nucleic Acids. |
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| Publication Number | Publication Date |
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| SE8600011D0 SE8600011D0 (en) | 1986-01-02 |
| SE8600011L SE8600011L (en) | 1986-07-03 |
| SE463212B true SE463212B (en) | 1990-10-22 |
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ID=8520136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SE8600011A SE463212B (en) | 1985-01-02 | 1986-01-02 | METHOD FOR IDENTIFYING NUCLEIC ACIDS |
Country Status (21)
| Country | Link |
|---|---|
| JP (1) | JPH0669400B2 (en) |
| AT (1) | AT397514B (en) |
| AU (1) | AU561382B2 (en) |
| BE (1) | BE903937A (en) |
| CA (1) | CA1271705A (en) |
| CH (1) | CH666696A5 (en) |
| DE (1) | DE3546312A1 (en) |
| DK (1) | DK164932C (en) |
| FI (1) | FI72146C (en) |
| FR (1) | FR2575493B1 (en) |
| GB (1) | GB2169403B (en) |
| HU (1) | HU196453B (en) |
| IE (1) | IE58290B1 (en) |
| IL (1) | IL77489A (en) |
| IT (1) | IT1201514B (en) |
| LU (1) | LU86238A1 (en) |
| NL (1) | NL189427C (en) |
| NO (1) | NO166743C (en) |
| RO (1) | RO94651B (en) |
| SE (1) | SE463212B (en) |
| ZA (1) | ZA859895B (en) |
Families Citing this family (36)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5288609A (en) * | 1984-04-27 | 1994-02-22 | Enzo Diagnostics, Inc. | Capture sandwich hybridization method and composition |
| US6060237A (en) * | 1985-02-26 | 2000-05-09 | Biostar, Inc. | Devices and methods for optical detection of nucleic acid hybridization |
| ES8707343A1 (en) † | 1985-06-13 | 1987-07-16 | Amgen | Method for performing nucleic acid hybridization assays. |
| US4868105A (en) * | 1985-12-11 | 1989-09-19 | Chiron Corporation | Solution phase nucleic acid sandwich assay |
| US4910300A (en) * | 1985-12-11 | 1990-03-20 | Chiron Corporation | Method for making nucleic acid probes |
| US4882269A (en) * | 1985-12-13 | 1989-11-21 | Princeton University | Amplified hybridization assay |
| FI76119C (en) * | 1986-02-27 | 1988-09-09 | Orion Yhtymae Oy | Quantitative determination of nucleic acid molecules and reagent packaging used in the process |
| AU622104B2 (en) * | 1987-03-11 | 1992-04-02 | Sangtec Molecular Diagnostics Ab | Method of assaying of nucleic acids, a reagent combination and kit therefore |
| WO1988010313A1 (en) * | 1987-06-26 | 1988-12-29 | E.I. Du Pont De Nemours And Company | Affinity removal of contaminating sequences from recombinant cloned na using capture beads |
| ATE112804T1 (en) * | 1987-07-31 | 1994-10-15 | Gen Probe Inc | POLYNUCLEOTIDE ASSAY USING OLIGONUCLEOTIDES TO ELIMINATE UNDESIRABLE CROSS-REACTIONS. |
| EP0305145A3 (en) * | 1987-08-24 | 1990-05-02 | Ortho Diagnostic Systems Inc. | Methods and probes for detecting nucleic acids |
| EP0304845A3 (en) * | 1987-08-28 | 1991-03-06 | Profile Diagnostic Sciences Inc. | Method and kit for assaying gene expressions |
| CA1325761C (en) * | 1987-12-25 | 1994-01-04 | Takanori Oka | Method of detecting an intended nucleic acid in a sample |
| DE3800644A1 (en) * | 1988-01-12 | 1989-07-20 | Boehringer Mannheim Gmbh | PROCESS FOR HIGHLY SPECIFIC DETECTION OF NUCLEIC ACIDS IN SOLUTION |
| US5104791A (en) * | 1988-02-09 | 1992-04-14 | E. I. Du Pont De Nemours And Company | Particle counting nucleic acid hybridization assays |
| WO1989010979A1 (en) * | 1988-05-10 | 1989-11-16 | E.I. Du Pont De Nemours And Company | Process for rapid nucleic acid detection |
| US5185243A (en) * | 1988-08-25 | 1993-02-09 | Syntex (U.S.A.) Inc. | Method for detection of specific nucleic acid sequences |
| US5858652A (en) * | 1988-08-30 | 1999-01-12 | Abbott Laboratories | Detection and amplification of target nucleic acid sequences |
| AU629845B2 (en) * | 1988-08-30 | 1992-10-15 | Abbott Laboratories | Detection and amplification of target nucleic acid sequences |
| GB2225112A (en) * | 1988-11-22 | 1990-05-23 | Ici Plc | Hybridisation probes |
| US5082935A (en) * | 1988-12-15 | 1992-01-21 | Amoco Corporation | Diagnostic reagents made by attaching cytidine containing nucleic acid probes to amino functionalized solid supports by bisulfite mediated transamination |
| CA2025236A1 (en) * | 1989-02-06 | 1990-08-07 | Walter King | Probes and methods for the detection of listeria |
| AU5269590A (en) | 1989-03-10 | 1990-10-09 | Gene-Trak Systems | Immobilized oligonucleotide probes and uses therefor |
| ATE155821T1 (en) * | 1989-03-10 | 1997-08-15 | Vysis Inc | INTERFERENCE PREVENTION USING AFFINITY CAPTURE SCHEMES |
| JPH05501052A (en) * | 1989-07-11 | 1993-03-04 | マイクロプローブ・コーポレーション | Quantification of bacteria using a nucleic acid hybridization assay |
| US5334501A (en) * | 1989-07-11 | 1994-08-02 | Microprobe Corporation | Quantification of bacteria using a nucleic acid hybridization assay |
| GB8924989D0 (en) * | 1989-11-06 | 1989-12-28 | Scotgen Ltd | Method and device for the detection of antibiotic resistance |
| US5580970A (en) * | 1989-12-01 | 1996-12-03 | Amoco Corporation | Detection of HPV transcripts |
| AU7429591A (en) * | 1990-04-18 | 1991-10-24 | Gene-Trak Systems | Nucleic acid probes for the detection of giardia lamblia |
| WO1992015708A1 (en) * | 1991-02-27 | 1992-09-17 | Amoco Corporation | Methods for improving the sensitivity of hybridization assays |
| WO1993020234A1 (en) * | 1992-03-31 | 1993-10-14 | E.I. Du Pont De Nemours And Company | A rapid, high capacity nucleic acid based assay |
| US7713528B1 (en) | 1993-02-18 | 2010-05-11 | Enzo Therapeutics, Inc. | Method for in vivo delivery of active compounds using reagent conjugate |
| US5853993A (en) * | 1996-10-21 | 1998-12-29 | Hewlett-Packard Company | Signal enhancement method and kit |
| GB0016833D0 (en) | 2000-07-07 | 2000-08-30 | Lee Helen | Improved dipstick assays (2) |
| US7465540B2 (en) | 2000-09-21 | 2008-12-16 | Luminex Corporation | Multiple reporter read-out for bioassays |
| CN1303221C (en) * | 2003-01-27 | 2007-03-07 | 英科新创(厦门)科技有限公司 | Method for detecting target nucleic acid using affinity amplifying integrated signal group |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI63596C (en) * | 1981-10-16 | 1983-07-11 | Orion Yhtymae Oy | MICROBIA DIAGNOSIS FOERFARANDE SOM GRUNDAR SIG PAO SKIKTSHYBRIDISERING AV NUCLEINSYROR OCH VID FOERFARANDET ANVAENDA KOMBINATIONER AV REAGENSER |
| FR2518755B1 (en) * | 1981-12-23 | 1986-04-11 | Pasteur Institut | PROBE CONTAINING MODIFIED NUCLEIC ACID RECOGNIZABLE BY SPECIFIC ANTIBODIES AND USE OF THE SAME TO DETECT AND CHARACTERIZE A HOMOLOGATED DNA SEQUENCE |
| GB8306426D0 (en) * | 1983-03-09 | 1983-04-13 | Malcolm A D B | Detecting polynucleotide sequence |
| CA1228811A (en) * | 1983-05-05 | 1987-11-03 | Robert G. Pergolizzi | Assay method utilizing polynucleotide sequences |
| NO843838L (en) * | 1983-09-26 | 1985-03-27 | Ortho Diagnostic Systems Inc | PROCEDURE FOR DETECTING NUCLEIC ACID |
| ZA849594B (en) * | 1983-12-12 | 1985-08-28 | Miles Lab | Nucleic acid hybridization assay employing antibodies to intercalation complexes |
| FR2558172B1 (en) * | 1984-01-16 | 1986-06-13 | Inst Nat Sante Rech Med | PROBE CONTAINING MODIFIED NUCLEIC ACID AND RECOGNITION BY SPECIFIC ANTIBODIES AND USE OF SUCH PROBE AND THESE SPECIFIC ANTIBODIES TO DETECT AND CHARACTERIZE A HOMOLOGATED DNA SEQUENCE |
| CA1223222A (en) * | 1984-02-22 | 1987-06-23 | Nanibhushan Dattagupta | Immobilized nucleic acid-containing probes |
| FR2560298B1 (en) * | 1984-02-28 | 1988-07-15 | Mors | METHOD AND DEVICE FOR ADJUSTING THE PRECISION OF THE PRESSURE DROP OF A FLUID INITIALLY AT HIGH PRESSURE, SUCH AS A HYDRAULIC FLUID FOR EXAMPLE SUPPLYING A HYDRAULIC GROUP, AND APPLICATION OF THE METHOD TO THE DEVICE IN A HYDRAULIC GROUP OR PLANT INTEGRATED OR NOT, ESPECIALLY IN A MACHINE TABLE OR ANY TOOL SUPPORT DEVICE |
| NZ211453A (en) * | 1984-03-22 | 1989-01-06 | Biotechnology Research Enterpr | Aryl azides, their preparation and use in the detection, localisation and isolation of polynucleotides |
| US4766062A (en) * | 1984-05-07 | 1988-08-23 | Allied Corporation | Displacement polynucleotide assay method and polynucleotide complex reagent therefor |
-
1985
- 1985-01-02 FI FI850004A patent/FI72146C/en not_active IP Right Cessation
- 1985-12-17 CH CH5378/85A patent/CH666696A5/en not_active IP Right Cessation
- 1985-12-20 GB GB08531414A patent/GB2169403B/en not_active Expired
- 1985-12-24 BE BE0/216059A patent/BE903937A/en not_active IP Right Cessation
- 1985-12-27 NO NO855308A patent/NO166743C/en not_active IP Right Cessation
- 1985-12-27 JP JP60299797A patent/JPH0669400B2/en not_active Expired - Lifetime
- 1985-12-29 HU HU855030A patent/HU196453B/en unknown
- 1985-12-30 RO RO121637A patent/RO94651B/en unknown
- 1985-12-30 IT IT23408/85A patent/IT1201514B/en active
- 1985-12-30 DE DE19853546312 patent/DE3546312A1/en active Granted
- 1985-12-30 FR FR858519394A patent/FR2575493B1/en not_active Expired - Lifetime
- 1985-12-30 LU LU86238A patent/LU86238A1/en unknown
- 1985-12-30 ZA ZA859895A patent/ZA859895B/en unknown
- 1985-12-30 AT AT0376785A patent/AT397514B/en not_active IP Right Cessation
- 1985-12-31 CA CA000498834A patent/CA1271705A/en not_active Expired - Lifetime
- 1985-12-31 NL NLAANVRAGE8503597,A patent/NL189427C/en not_active IP Right Cessation
- 1985-12-31 AU AU51748/85A patent/AU561382B2/en not_active Expired
- 1985-12-31 IE IE333385A patent/IE58290B1/en not_active IP Right Cessation
- 1985-12-31 IL IL77489A patent/IL77489A/en not_active IP Right Cessation
-
1986
- 1986-01-02 DK DK000386A patent/DK164932C/en not_active IP Right Cessation
- 1986-01-02 SE SE8600011A patent/SE463212B/en not_active IP Right Cessation
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