CA2073989A1 - Immobilization of nucleic acids - Google Patents
Immobilization of nucleic acidsInfo
- Publication number
- CA2073989A1 CA2073989A1 CA002073989A CA2073989A CA2073989A1 CA 2073989 A1 CA2073989 A1 CA 2073989A1 CA 002073989 A CA002073989 A CA 002073989A CA 2073989 A CA2073989 A CA 2073989A CA 2073989 A1 CA2073989 A1 CA 2073989A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- labelled
- detectably
- nucleotides
- hybrid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
Abstract
A b s t r a c t Method for the detection of nucleic acids in which a nucleic acid probe having two or more nucleotides modified by immobilizable group which are not directly adjacent in the nucleotide sequence is used.
Description
.I~m bili~ati~ o~ n~L io aai4~
The invention concerns a method for detecting nucleic acids, the use of special nucleic acids to immohilize nucleic acid~ and a system ~or tha detection of nucleic acids.
The immobili~ation of nucleic a~ids is important in many areas for example when separating nucleic acids from other m~tarials, when i~olating nucleic acids with a special nucleotide sequence from a nucleic acid mixture and in additiDn for the detaction Gf nucleic acids ~nucleic acid diagnostics). In all these areas utilization is made of the special affinities o~ nucleic acids to th~e nucleic acids which have ~ nucleotide ~equence which i8 essentially complementary to them and can hybridize under certain conditions with the desired nucleic acids. The complementary nucleic acids which are used for this which are denoted capture probe in the following are usllally bound to a solid phase e.g. a membrane, a gel, a bead or a tube. Recently it has been established that the immobilization of nucleic acids to solid phases via covalently bound capture probes ha~
drawbacks in several respects e.g. the rate of immObiliZatlOn i8 reduced. Therefore it was suggested that the capture probe be specifically bound to the solid phase via affinity bindingO Such a binding for example via antibodies/haptens or biotin/(strept-) avidin) enables a hybridi~ation of the nucleic acid with the capture probe in a liquid ancl subsequent - 2 ~ ~ ~73~Y
immobilization of the hybrid formed. such a procedure is described for example in EP-A-O 139 489.
The ob;ect of the pre~ent inventlon was to increase the effectiveness o~ the immobilization in order, in particular, to obtain more sensitive tests for nucleic acids.
The inv~ntion conaern~ a method for the detection of an analyte hucleic acid which comprises the following steps: `
- Hybridization of a detectahly labelled nucleic acid or of a detectably labelled nucleic acid hybrid with an immobilizable nucleic acid probe;
- i~mobilization of the hybrid formed and - detection of the amount of immobilized hybrid via the amount of label, wherein the i~nobilizable nucleic acid prob~
contains two or more nucleotldes modified by immobilizable groups which are not directly adjacent in the nucleotide sequence.
The invention als~ concerns the use of the nucleic acids mentioned in the method as well as a system for detecting nucleic acids.
An analyte nucleic acid within the sense of the invention is a nucleic acid which is to be detected~ It is usually present in a sample which also contains further components, for example a tissue or a liquid. In - 3 _ t~r~
particular th~re are ~lso additlonal nucleic acids in the sampl~ which are not intended to be detected. The nucleic acid can be any type of nucleic acid r for example DN~, RN~ or ~ragments thereoP. I~ the analyte nucleic acid is originally present in the sample in a double-stranded form it is pre~erably converted into the single~stranded form. I~ the analyte nucleic acid is pre~ent bound to a solid phase it is preferably brought into ~olution.
A detectably labelled nucleic acid is understood as a nucleic acid whose presence or amount can be determined with the aid of a detection system. For this purpose a detectably labelled nucleic acid has modifications as compared to a normal nu~leotide seguenoes. Suitable modifications are familiar to one skilled in the art. An example o~ a modification are chemical groups which are not normally present in nucleic acids such as enzymes, coloured or ~luorescent molecules and ligands, pre~erably residues capable of binding in~unological.ly.
Examples o~ the latter are in particular haptens such as digoxigenin and also vitamins such as biotin. In the case of enzymes the detection is usually carried out via a colour-forming reaction catalyzed by the enzyme.
Coloured or fluorescent molecules can be detected directly by photometric or fluorometric means. Ligands are ~sually contacted with a corresponding enzyme-labelled or dye-labelled receptor which has affinity to the ligands and are d~tected by this means.
A detectably labelled nucleic acid hybricl i5 understood as a partially double-stranded hybrid of at least two mlcleic acids. In this case either both nucleic acids or only one of them may be detectably labelled as described above. It is important that the nucleic acid hybrid has ~ ~ r~
a sinyle-stranded region. In a so-called sandwich test the hybrid preferably consists of the (non l~belled) analyte nucleic acid and a detectably labelled detector probe.
An immobilizable nucleic acid probe is understood as a nucleic acid probe which is modified by a residue which is not presenk in the usual nucleic acidsu Su~h residues are residues which have an affinity to another material.
Examples are partners of a biospecific reaction between a ligand and a receptor for example an immunological reaction, a reaction between sugar and lectin or between vitamin and binding protein. Particularly preferred residues are haptens or biotin. In the method accorcling to the present invention the residue on the immobilizable nucleic acid probe is di~fererlt from the det~ctable residue of the detectably labelled nucleic acid.
The steps of the method of detection describPd in the following are known as indlvidual steps or can be carried out analogou~ to known m~th~ds. Reference ~g in particular made to Molecular Cloning, Editor Sambrook et al., CSH 1989. These also include the known methods for the production of labelled nucleoside triphosphates, the chemical synthesis of modified ~nd unmodified oligonucleotides, the choice of hybridization conditions by which means a specificity can be achieved which i.s dependent on the extent of homology between the nucleic acids to be hybridized, their GC content and their length, as well as the formation of nucleic acids from nucleoside triphosphates with the aid of polymerases and if desirsd, using so-called primers. An essential feature of the present invention is the use of a special capture probe.
2 ~ 3 ~43 In a first step of the method according to the present invention a detectably lahelled nucleic acid or a detectably labelled nucleic acid hybrid is produced using the analyte nucleic acid. This can for example be carried out by detectably labelling the analyte nucleic acid itself. This can be achieved by the enzymatic incorporation of labelled nucleoside triphosphates or by elongating the analyte nucleia acid with labelled nucleoside triphosphates (tailing). A furthex possibility is to amplify a region of the analyte nucleic acid whila incorporating detectably labelled mononucleoside triphosphates or detectably labelled oligonucleotides. Many methods are known for this from the state of the art.
One possibility is to carry out an amplification ~y m~an~ of the pol~mera3e chain reaction aacording to EP-A-0 200 362 using the analyte nucleic acid as the template nucleic acid. In this case two primers are used one of which is complementary to a region of the analyte nucleic acid and the other is complementary ko a part of the oppo~ite strand of the analyte nucleic acid to form a multitude of copies of the strand and opposite strand of the analyte nucleic acid by elongation of the pri.mer on the template. In order to produce the detectably lab~lled nucleicc acid, at least one detectably labelled mononucleoside triphosphate is used in the elongation of the primer or a labelled primer is used.
A further possibility ar.ises from the use of the ligase chain reaction according to W0 90/06376 if at least one of the oligonucleotides is detectably labelled.
A further possibility is described in EP-A-0 329 822. In this process a DNA strand is formed which is - 6 - ~ Q 7 ~
complementary to substantial parts of the analyte nucleic acid and a primer containing a promoter is incorporated. After degradation of the analyte nucleic acid, a transcribable double strand is ~ormed which can be used to form a multitude of detectably labelled transcripts while incorporating labelled mononucleoside triphosphates.
The method according to the present invention has proven to be particularly efficient when in the form of a sandwich nucleic acid assay e.g~ according to EP-A-O 192 168. The method according to the present invention differs from ~he ~nown sandwich assays in that a particular type of capture probe is used.
In a ~urther step the detectably labelled nucleic acid or the detectably labelled nucleic acid hybrid is brought into contact with an immobllizable nucleic acid probe under hybridization conditions.
The immobilizable nucleic acid probe according to the present invention contains two or more nucleotides modified by immobili~able groups which are not directly adjacent in the nucleotide sequence. Apart from the number of ligands (reporter groups) the distance between the ligands is crucial and should be preferably 10 or more nucleotides in order to observe this efEect. For example oligodeoxyribonucleotide probes which contained 5 biotin residues (biotin corresponds to the ligand) coupled in direct succession and were used as capture probes to bind an analyte to a streptavidin matrix did not result in an increase in the sensitivity in the total assay compared to a reference probe which was only linked to one biotin residue ~see example 2). The "capture probe" can ~e an oligonucleotide or a 7 ~ ;3~j~S
polynucleotide (DNA ~r R~) which can hybridlze in a suitable manner with the analyte nucleic acid to be detected. It can be single-stranded (e.g.
oligodeoxyribonucleotide, oligoribonucleotide) or double-stranded ~e.g. plasmid, fragment). In the latter case the capture probe has to be denatured be~ore hybridization with the analyte. A particularly preferred embodiment utilizes nucleic acid probes in which the terminal nucleotide~ of the nucleic acid probe are modified in each case.
An oligodeoxynuc}eotide is preferred which has a length between ll and 40 nucleotides whereby the label is appropriately attached to the 3'- and 5'- end in ordler to meet the requirement for distance as described above.
The immobilizable residues can in principle be bound to the base part or to the ~ugar part or to the phosphate part of the nucleotides. Such immobilizable nucleic acid probes can be produced acaording to known method~ or analogous to known methods. The attachment of a ligand to nucleotides with modi~ied bases is described for example in Nucleic Acids Res. 15 ~12), p. 4857 to 4876 ~1987); Nucleic Acids Res. 16 (9), p. 4077 to 4095 (1988); Nucleic Acids Res. 17, p. 4643 to 7650 (1989) or can be carried out by introducing a modified nucleotide which already contains the ligand (e.g. Nucleic Acids Res. 18 (15), p. 4355 to 4360 (1990). A ~urther suitable chemical method for incorporating several primary amino groups in synthetic oligonucleotides which can then be used for binding to an immobilizable residue is described in Nucleic Acids Res. 17 (18), p. 7179 to 7194 ~1989). The attachment o~ the ligand to a nucleic acid can also be carried out by a bisulfide-catalyzed transamination (Nucleic Acids Res. 12 (2), p. 989 to - 8 - ~f~
1002 (1984)). The attachment of several immobilizable xesidues can al60 be carried out by enz~matic methods e.g. by tailing using a nucleoside triphosphate containing the appropriate residue tDNA 5 (4), p. 333 to 337 (1986~) or by random priming (Analyt. Biochem~ 132, p. 6 lg~3)~
Chemically synthesized oligonucleotides are particularly preferred within the scope of the present invention since they hava the advantage that the distance between two modified nucleotides can be exactly determined bef orehand.
The use of multiply labelled nucleic acids as detection probe~ was known previously. For example in EP-A-0 330 221 an oligonucleotide or a polynucleotide is labelled with biotin and subse~uently biotin is detected by means o~ a streptavidin-enzyme complex. The separation of two ~iotin-dUMP residues in the nucleotide sequence by simultaneous incorporation of dTTP is de~cribed as being more of a disadvantage. In Nucleic Acids Res. 1~ (15), p~ 4358 it is shown that by attaching several immobilizable residues to nucleic acids their binding to solid phases can be improved.
In Nucleic Acids Res. 18, p. 4345 to 4354 (1990) multiply labelled nucleic acids are also used as a detection probe. The production of multiply labelled nucleic acids in which the label is attached to the phosphate residue .is described in W0 90/08838.
If the sample contains analyte nucleic acid, a nucleic acid hybrid forms from the detectably labelled nucleic acid or the detectably labelled nucleic acid hybrid and - 9 - l~v ~
the immobilizable nucleic acid probeO In the m~thod according to the present invention this is bound via the immobilizable residue to a solid phase. The surface of the solid phase contains groups which have an affinity to the immobilizable residue o~ the nucleic acid probe, for example a receptor. If the immobilizable group is a hapten, the solid phase preferably contclins an antibody against this hapten. In the case of biotin the solid pha~e contains a biotin binding protein such as avidin or streptavidin.
The amount of immobilizable hybrid is a measure for the amount or the presence of the analyt~ nucleic acid. This amount can be detscted via the amount of label which is immobilized on th~ solid phase. This is preferably carried out in a known manner and depends on the type of label used. Before carrying out the detection reaction, the solid phase is preferably removed from the liquid phase. This at the same time results in the removal of the starting material ~or the production o~ the detectably labelled nucleic acid or for the detectably labelled nucleic acid hybrid or of an excess detec~ably labelled nualeic acid probe in the case of a sandwich hybridization togather with the li~uid. The detection of a nucleic ac~d by the method according to the present invention, especially if the test is to be carried out quantitatively, encompasses comparing the measurement signal which was obtained with the sample of unknown analyte nucleic acid content with the measurement signal or measurement signals which were obtained with one or several samples with known analyte nucleic acid content.
The use o~ a calibration curve is preferred which can also be provided in the form of entered data.
1 0 ;~ ?~ (.J ~
The invention in addition concerns the use of a nucle.ic acid which contains two or more nucleotides modified ~y immobilizable groups which are not directly adjacent in the nucleotide sequence ~or immobilizing nucleic acids.
This aspec~ is based on the ~act that the nucleic acid probes according to the present invention guarantee a ~urprisingly improved immobilization~ T~he method can for example be used in the affinity separation of nucleic acids.
The invention also relates to a system for the detection of nucleic acids which contains one or several nucleic acids containing two or more nucleotides modified by immobilizable groups which are not directly adjacent in their respective nucleotide sequence and at least one solid phase which has a specific af f inity to the lmmobllizable groups of tha nucleic acid. Be~ore starting a detection method it is pr~ferable that the nucleic acids and the sclid phase are present separated ~rom one another in the system. ~n addition the system can also contain further components which are necessary or helpful for the detection of nucleic acids~ These in particular include pH buffers and reagents for detecting the label.
Fig. 1 shows a diagram of the binding of an analyte nucleic acid A via a capture probe F according to the present invention to a solid phase coated with streptavidin S in a sandwich test. The detectably (represented by a rhombus) labelled detector probe is denoted D.
Fig. 2 shows the binding to the wall of a labelled analyte nucleic acid A' via a capture probe according to the present invention to a solid phase coated with streptavidin S.
Fig. 3 illustrates the increase of the san~itivity of a sandwich nucleic acid test in which thQ immobilizable groups are located on adjacent nucleotide6 by separating the groups according to the present invention. It is clear that the increa~e in sensitivity when using only two biotin residue~ which are each at t:he ends of the oligonucleotide is unexpectedly even more than when 10 biotin residues are used at one end ~see also example 2).
Fig. 4 shows the increase in sensitivity in the sandwich test when using tws biotin residues compared to the use of one biotin resldue with an oligonucleotide length of 30 nucleotides.
Fig. 5 shows the comparison of sensitivity of Figure 4, but with oligonucleotides having a length o~ 20 nt.
Fig. 6 shows the compari~on of sen~itivity for a method in which an analyte nucleic acid wa5 amplified and at the same time labelled by PCR and in which nucleic acid probes with a length of 15 nucleotides and having one or two biotin residues were used as the capture probe.
12 - ~ Q 7 3 9 ~ ~
The curves shown in figures 4 to 6 can also be used as calibration curves for determining an unknown a~ount of an analyte nucleic acid.
Fig. 7 shows the formula for the aminomodifier II.
The invention is elucidated in more detail by the following example~.
- 13 ~ ,~J,~
PrQduction of oli~onucleot~des A: Explanatory notes All oligonu~leotides were produced with the aid of a DNA synthesizer 8700 from the Biosearch Company using the "phosphoramidite method" published by Caruther~ et alO Methods Enzymol. 154, 287, 1987.
The ~-cyanoethyl~nu~leoside phosphoramidites used were obtained ~rom Roth ~Karlsruhe, GFR), D
biotino~l-aminocaproic acid-N-hydroxysuccinimide eq~ter was from Boehringer Mannheim ~Mannheim, GFR), aminomodifier 2 (AM II) from Beckmann Instrumen~s GmbH ~Fullerton, CA, USA). All other reagents and solvents were used in the best ~uality available.
The purification of the biotlnylated oligonucleotide~ was carried out by HPLC (Spectra Physics, Darmstadt, GFR) using a Mono Q HR 5/5 ion-exchange column from Pharmacia (Freiburg, GFR) or a Cl~ column (LiChrosorb RP 18-5, CS-Chromatographie Service, Langerwehe, GFR). Spectra/Por lO00 membranes (Roth, Karlsruhe, GFR) were used for the dialysis.
B) Synthesis ~ad ~ri~ication of t e biotlnvlated oli~onucleotides The biotinylated oligonucleotides were all prepared in the Biosearch synthesizer on a 1 ~mol scale using the trityl-off as well as cleave-off programme. Since there was no carrier material available with aminomodifier 2, a th~midine carrier was used and subsequently aminomodifier 2 (see Fig.
7) was condensed to this according to the standard coupling program ~0.1 M in acetonitrile).
A~er the amlnomodifier, a further th~midine was coupled to the 5' end in order to protect the primary hydroxyl group of the aminomodi.~ier ~rom attack during the ammonia treatment and the glyceryl residue from being again partially cleaved o~f. A~ter completion of the .~ynthesis and cleavage of the protecting groups with concentrated NH3 solution at 55C/5h, all solvents were removed in a vacuum, the residue was taken up in water and the product was purified by ion-exchange chromatography (Mono ~ HR 5~5; A~ 0.25 mM Tris/HCl, 0.3 M NaCl; ~:
0.25 Tris/HCl, 1 M MaCl; in 60 min from 0 to 100 B, flow 1 ml/min). Subsequently the oligodeoxynucleotide was desalted by dialysis (Spectra/Por 1000, Roth). Afterwards the oligonuclotides were biotinylated by taking up 5-10 O.D.260nm of the oligodeoxynucleotides in 0.5 ml 0.05 M K2HP04/KH2P0~ buffer and adding a solution of 5 mg D-biotinoyl-aminocapxoic acid-N-hydroxysuccinimide ester in 0.5 ml DMF. The reaction mixture was incubated overnight at 37C, subsequently the solvent was removed in a vacuum, the residue was taken up in redistilled water and - 15 ~ a~
excess biotin was removed by filtration. The further purification was carried out by means of reversed phase ~PLC with a gradient o~ ~: 0.1 M
triathylammonium acetate p~I 7; 5 % acetonitrile, B:
O.1 M triethylammonium acetate pH 7; 40 %
acetonitrile. The gradient was run ~rom 20 % B to 80 % B within 40 min at a flow rate of 2 ml/min.
The dif~erence in the ret~ntion time between the twice biotinylated oligonucleotides and the corresponding aminomodi~ier-modi~ied starting oligonucleotides was between 2 and 4 minutes depending on the length of the sequence. The biotinylated oligodeoxynucleotid2s could bs obtained in 1.5 to 3.5 O~D.2~0 after renewed dialysis.
~etaction o~ ~v analyte D~A in a sandwich form taklnq into account_the site o~. modification The test is carried out in a sandwich with binding of the analyte DNA to a solid phase. A partial sequence of the hepatitis B virus DNA which is present clon~d in a plasmid is used as the analyte DNA. The analyte DNA is bound to a streptavidin solid phase by means of a biotin-labelled oligonucleotide whose sequence is complementary to a region of the HBV DNA. The detection of the bound analyte DNA is then carried out by means o~
a diyoxigenin-labelled oligonucleotide which in turn is complementary to another region of the analyte DNA. This can be recognized by anti-digoxigenin antibodies which are conjugated with horseradish peroxidase and the hybrid is subsequently detected by an enzyme-catalyzed colour-forming reaction.
A dilution series of the plasmid in a range of 4~4 ~g/ml, 2.2 ~g/ml, 1.1 ~g/ml, 0.55 ~g/ml in TE buffer ~10 mM Tris-HCl, 1 mM EDTA, pH 7.5~ is prepared. In order to denature khe double-strande~ DNA, 10 ~1 ~ M
NaOH 16 added to 90 ~1 of the plasmid solution and i~cubated for 10 min at room temperatur~. 20 ~1 of the denaturation prepaxation i~ subse~uently pipetted into a well o~ a microtitre plate ~oated with streptavidin and immediately neutralized by addition of 180 ~1 hybridization solution ~50 mM Na phosphate buffer, 0.75 M NaCl, 0.075 ~ Na citrate, 0.05 % bovine ~erum albumin, pH 5.4). This results in the following concentration~ of the plasmid in the testo 5~, 100, ~00, 400 ng/ml. The hy~ridi~ation solution in addition contains 200 ng/ml of a digoxigenin-labelled oligonucleotide (labelled once with digoxigenin at the 5' end, position 287c-24~c in the HBV genome) and of a biot~n-labelled oligon~cleotide (position 2456c-2417c in the HBV genome). The te~t is carried out with oligonucleotldes havi~g different degrees of bio~in labelling:
HBV-Oli 7-4 (1 biotin, 40mer), SEQ ID NO 1:
5'-T(bio-AMII)-CATTGAGATTCCCGAGATTGAGATCTTCTGCGACGCGGCG-3' HBV-Oli 7-5 ~5 biotin, 40mer), SEQ ID NO 2:
5'-T-(bio-AMII)5-CATTGAGATTCCCGAGATTCAGATCTTCTGCGACGCGGCG-3' HBV-Oli 7-6 (10 biotin, 40 mer), SEQ ID NO 3:
5'T-(bio-AMII)10-CATTGAGATTCCCGAGATTGAGATCTTCTGCGACGCGGCG-3' 17 ~ /7 ;~ ~3 f~ J
HBV-Oli 7-7 (2 biotin, 40mer~, SEQ ID N0 4~
5'T-(bio MII)-cATTGAGATrrcccGAGATTcAGATcTTcTGcGAcGcGGcG
(AMII-bio)-T-3' The hybridization preparation i~ incubated for 3 h at 37C in the microtitre plate while shaking. A~t~r aspirating the solution it is washed 2 x 10 min with 0.3 M NaCl, 0.03 M Na citrate, 002 % Na dodecylsulate at 370c and ~ubsequently once ~or a short time at room temperature with 0.9 % NaCl in order to remove non-bound reaction partners from the test. 20 mU/ml o~ an anti-digoxigenin antibody-horseradish peroxidase conjugate in 103 mM Tris-HCl (p~ 7.5), o.s % NaCl, 1 ~ bovin~ serum albumin is added and incubated ~or 30 min at 37C while shaking. Non-bound con~uyate is removed by briefly washing three times with 0.9 % NaCl at room temperat.ure.
The deteotion r~action is started by addition of thQ
substrate solution ABTS~ ~.9 mM, 2,2'azino.di~[3-ethylbenzthiazoline sulfonic acid (6)3-dia~m~nium salt).
The incubation is carried out ~or 30 min at 37C while shaklng. The absorbance i8 subsequently measur~d at 405 nm by means of an E~ISA reader.
The results ar~ ~hown in Fig. 3.
~amp~ 3 Detection of HBV-analYte DNA in a sandwich format takinq into account the chain_len~th and the site oE
modification A d.ilution series of the plasmid which contains HBV-specific sequences is prepared in a range from 62.5 to 250 ng/ml in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. 10 ~1 5 M NaOH is added to 90 ~1 of the plasmid solution and incuhated for 10 min at room temperature~ 20 ~1 of this denaturation preparation is pipetted into a well of a microtitre plate coated with streptavidin and 180 ~1 hybridization solution (50 mM phosphate buffer, 0.75 M
NaGl, 0.075 M Na citrate, 0.05 ~ bovine serum albumin (~SA), pH 5.4) is added. 200 ng/ml o~ the biotin-labell~d captux~ oligonucleotide and of th~ digoxigenln-labelled oligonucleotides ~pos. 405-444 in the HBV
genome, 40mer, labelled twice with digoxigenin at the 3' and 5' end) are added to the hybridization solution.
Thus the analyte DNA concentration in the test is 6.25, 12.5, 25 and 50 ng/ml.
HBV-Oli 25-3 ~pos. 2327-2356, 30mer, 1 biotin), SEQ ID NO 5:
5'-T-(bio-AMII)-CACTTCCGGAAACTACTGTTGTTAGACGAC-3' H~V-Oli 25-4 (pos. 2327-2356, 30mer, 2 biotin), SEQ ID NO 6:
5'-T-(blo-AMII~-CACTTCCGGAAACTACTGTTGTTAGACGAC-~MII-bio-~-T-3' HBV-Oli 33-1 (pos. 2332-2351, 20mer, 1 biotin).
SEQ ID NO 7:
5'-T-(bio-AMII)-CCGG~AACTACTGTTGTTAG-3' HBV-Oli 33-2 (pos. 2332-2351, 20mer, 2 biotin), SEQ ID NO 8:
5'-T-(bio-AMII)-CCGGAAACTACTGTTGTrrAG-(AMII-Bio)-T-3' ~he stated positions relate to the HBV genome.
- 19 - ~?~ ~ 3 ~
The hybridi.zation preparation is shaken for 3 h at 370C.
Subsequently it is washed 2 x lo min with 0.3 M NaCl, 0.03 M Na citrate, o.~ % Na dodecylsulfate at 37OC and then once briefly wi~h o.s % NaCl at room temperature.
Su~sequently 20~ ~l of the anti-digoxigenin antibody-horseradish peroxidase conjugate (200 mU/ml in 100 mM
Tris-HCl, pH 7.5 9 0 . 9 % NaCl, 1 % BSA) is added and incubated for 30 min at 370C whil2 shaking. After washing again three times with O . 9 % NaCl, the substrate solution ~1.9 mM ABTSR) is pipetted shaken again for 30 min at 370C and th~n the absorbance is measured at 405 nm by mean6 of an ELISA reader.
The result~ ~or the 30 nt oligonucleotid~s are shown in Fig. 4, those for the ~o nt oligonucleotides are shown in Fig. 5.
B~mpla Detection of_~Ç~ Products wit~ nqlY=_and_~ e-lahelled oli~onucleotides For the DNA test for the hepatitis B viruses, the viral DNA is firstly amplified by means of the polymerase chain reaction (EP~A-0 200 362). During this amplification digoxigenin is incorporated into the DNA
in the form of Dig~ dUTP (EP-A-32~474). Subsequently this hapten allows a sensitive detection of the PCR
product in a heterogeneous immunoassay. The binding of the digoxigenin-labelled DNA to the walls of the streptavidin-coated tubes is carried out by means of a biotin-labelled oligonucleotide who~e region is complementary to a region of the PCR product. The test is again carried out using an anti-digoxigenin antibody-~ ~ ~ 3 ~ . ~
~ 20 ~
horseradi~h peroxidase conjugate and a sub~e~uent colourreaction.
HBe-positive human plasma with a virus titre of 1 x 101 hepatitis B viruses per ml is diluted in normal serum so that the virus content in the dilutions is 1 x 107 viruses/ml. For the lysi~, 10 ~l 0.2 M NaOH is added to 10 ~l of the virus dilution and incubatisd for 1 h at 37C. The lysis preparation i6 neutrali2ed by addition of 30 ~l neutrali~ation mixture (100 mM KCl, 50 mM Tris-HCl, pH 6.5, 3 mM MgCl2). 20 ~l of this solution is used in the subsequent amplification reaction (amplification conditions: 200 nM of each of the PCR prim~rs, 200 ~M
each of dATP, drTp/ dGTP, 175 ~M dTTP, 25 ~M
digoxigenin~ 2'-dUTP, 2.5 U Thermus aquQticus DNA
polymerase in 50 mM KCl, 10 mM Tris-HCl, pH 8.9, 1.5 mM
MgCl2, 0.01 % gelatin; total volume 100 ~l). The preparation is covered with a layer of 100 ~l mineral oil and incubated ~or 30 cycles in a thermo-cycler (Perkin-Elm~r):
30 sec 92C, 30 sec 50C, 60 sec 70C.
Due to the position of the PCR primers (PCR primer 1:
position 1937-1~60; PCR primar 2: position 2434c-2460c in the HBV genome) a 500 bp long DNA fragment is produced which is subsequently detected in a two-component test system in streptavidin-coated microtitre plates.
The PCR preparation is diluted in H20 in such a way that in relation to the original virus concentration of 1 x 107 viruses per ml serum, dilutions ar~ made which correspond to a range of 1 x 105 to 1 x 104 viruses/ml.
20 ~1 of the diluted PCR preparatlon is incubated in a ~inal concentration of 0.1 N NaO~ in a ~olume of 50 ~1 for 10 min at room temperature for the denaturation.
20 ~1 of the denaturation solution are pipetted together with 180 ~1 hybridization solution (1 M NaCl, 0.~ M Na citrate, 67.5 mM Na phosphate, 0.05 % BSA, p~ 6.7) into a ~treptavidin-coated well of a microtitre plate. The biotin-labelled oligonuoleotides are added at a concentration of 100 ng/ml to the hybridization buffer:
HBV-Oli 34-1 (pos. 2299-2313, 15mer, 1 biotin) r SEQ ID NO 9:
5'-T-(~MII-bio)-AGACCACCAAATGCc~3' ~BV-Oli 34-2 (pos. ~299-2313, 15mer, 2 biotin) SEQ ID NO 10~
5' T-(AMII-bio)-AGACCACCAAATGCC-(bio-AMIIH)-T-3' The hybridization preparation i5 incubated for 3 h at 37C while skaking. Subsequently the ~olution is aspirated and the well is washed three time~ with 0.9 %
NaCl. 200 ~1 of a solution of 200 mU/ml anti-digoxigenin antibody-hor~eradlsh peroxidase con~ugate in 100 mM
Tri~-HCl, pH 7.5, 0.9 % NaCl, 1 % BSA is added and incubated for 30 min at 37C. After washing again with 0.9 % NaCl the substrate reaction is started by addition of 200 ~1 ABTSR. The photometric measurement is carried out after 30 min at 405 nm.
The results are shown in E'ig. 6.
~o~uerlce Protocol SEO ID ~0 1 ~ength of sequence: 41 bases Type of sequence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position- HBV genome 2456c-2417c (Hpbadw-data bank~
Antisense Modification N: T on this is bound to aminomodifier II via 3'-0 biotin 5'-N CAT TGA GAT TCC CGA GA~ TGA GAT CTT CTG CGA CGC GGC
G-3' SEQ I~ NO_2 Length of sequence: 41 base~
Type o~ sequence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2~56c-2417c Antisense Modification N: T on this is bound to 5 aminomodifiers II via 3'-0 5 biotin 5'-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
G-3' 2 ~
Length of sequence: ~1 bases Typ~ of saquence: nucleotide se~uence Type of strand: single strand Topology: linear Type of moleculeo part of genome DNA with modifi~ations N
Position: HBV genome 2456r-2417c Antise~se Modification N: T on this is bound to 10 aminomodifiers II via 3'-0 10 biotin 5l-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
G~3' Length of se~uence- 42 base~
Type of sequence: nucleotide sequence Type of st~and: single strand Topology: linear T~pe of molecule: part of genome DNA with modifications N
Position: HBV genome 2456c-2417c Antisense Modification N at T on this is bound to the 5' end: aminomodi~ier II via 3l_0 biotin Modification N at T on this is bound to the 3' end: aminomodifier II via 5' 0 biotin 5'-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
N-3' - 2~ -. f SE QID NO_5 Length of sequence: 31 basQs Type of se~uence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2327-2356 Modification N: T on this is bound to aminomodifier II via 3'-0 biotin 5'-N CAC TTC CGG AAA CTA CTG TTG TTA ~AC GAC 3' SE ~ID NO 6 Length o~ ~equence: 32 bases Type of sequence: nucleotide sequence Type of strand: single ~trand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2327-2356 Modi~ication N at T on this is bound to the 5~end: aminomodifier II via 3'-0 biotin Modification N at T on this i.s bound to the 3'end aminomodifier II via 5'-0 biotin 5'-N CAC TTC CGG AAA CTA CTG TTG TTA GAC GAC N-3' Len~th of sequence: 2 O bases Type of sequenceO nucleotide sequence Type of strand- ~;ingle strand Topology: linear Type of molecule: part Q~ genome DNA with modi~ications N
Position: H~Y genoma 2332-2351 Modi~ication N: T on this i~3 bound to aminomodif ier II via 3 ' -O biotin 5 '-NC CG(; A~A CTA CTG TTG TTA G-3 1 ~ ;.3 Length o~ sequence: 21 basPS
Type of sequence: nucleotide sequence Type o~ ~trand: single strand Topology: linear YPQ of molecule: part of genome DNA with modi~ication~ N
Position~ HBV genome 2332-2351 Modification N at T on this is bound to the 5' end: aminomodifier II via 3'-0 biotin Modification N at T cn this is bound to the 3' end: aminomodifier II via 5'-0 biotin 5'-NC CGG AAA CTA CTG ~TG TTA GN-3 SEQ ID NO ~
Length o~ sequence: 16 bases Type of sequence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2299-2313 Modification N: T on this is bound to aminomodifier II via 3'-0 biotin 5'-N AGA CCA CCA AAT GCC-3' SE~ ID NO 10 !.~
Length of sequence: 17 bases Type of sequence: nucleotide sequenc~
Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modi~ication6 N
Position: HBV genome 2299-2313 Modification N at T on this is bound to the 5' end aminomodifier II via 3'-0 biotin Modification N at T on this is bound to the 3' end: aminomodifier II via 5'-0 biotin 5'-N AGA CCA CCA AAT GCC N-3'
The invention concerns a method for detecting nucleic acids, the use of special nucleic acids to immohilize nucleic acid~ and a system ~or tha detection of nucleic acids.
The immobili~ation of nucleic a~ids is important in many areas for example when separating nucleic acids from other m~tarials, when i~olating nucleic acids with a special nucleotide sequence from a nucleic acid mixture and in additiDn for the detaction Gf nucleic acids ~nucleic acid diagnostics). In all these areas utilization is made of the special affinities o~ nucleic acids to th~e nucleic acids which have ~ nucleotide ~equence which i8 essentially complementary to them and can hybridize under certain conditions with the desired nucleic acids. The complementary nucleic acids which are used for this which are denoted capture probe in the following are usllally bound to a solid phase e.g. a membrane, a gel, a bead or a tube. Recently it has been established that the immobilization of nucleic acids to solid phases via covalently bound capture probes ha~
drawbacks in several respects e.g. the rate of immObiliZatlOn i8 reduced. Therefore it was suggested that the capture probe be specifically bound to the solid phase via affinity bindingO Such a binding for example via antibodies/haptens or biotin/(strept-) avidin) enables a hybridi~ation of the nucleic acid with the capture probe in a liquid ancl subsequent - 2 ~ ~ ~73~Y
immobilization of the hybrid formed. such a procedure is described for example in EP-A-O 139 489.
The ob;ect of the pre~ent inventlon was to increase the effectiveness o~ the immobilization in order, in particular, to obtain more sensitive tests for nucleic acids.
The inv~ntion conaern~ a method for the detection of an analyte hucleic acid which comprises the following steps: `
- Hybridization of a detectahly labelled nucleic acid or of a detectably labelled nucleic acid hybrid with an immobilizable nucleic acid probe;
- i~mobilization of the hybrid formed and - detection of the amount of immobilized hybrid via the amount of label, wherein the i~nobilizable nucleic acid prob~
contains two or more nucleotldes modified by immobilizable groups which are not directly adjacent in the nucleotide sequence.
The invention als~ concerns the use of the nucleic acids mentioned in the method as well as a system for detecting nucleic acids.
An analyte nucleic acid within the sense of the invention is a nucleic acid which is to be detected~ It is usually present in a sample which also contains further components, for example a tissue or a liquid. In - 3 _ t~r~
particular th~re are ~lso additlonal nucleic acids in the sampl~ which are not intended to be detected. The nucleic acid can be any type of nucleic acid r for example DN~, RN~ or ~ragments thereoP. I~ the analyte nucleic acid is originally present in the sample in a double-stranded form it is pre~erably converted into the single~stranded form. I~ the analyte nucleic acid is pre~ent bound to a solid phase it is preferably brought into ~olution.
A detectably labelled nucleic acid is understood as a nucleic acid whose presence or amount can be determined with the aid of a detection system. For this purpose a detectably labelled nucleic acid has modifications as compared to a normal nu~leotide seguenoes. Suitable modifications are familiar to one skilled in the art. An example o~ a modification are chemical groups which are not normally present in nucleic acids such as enzymes, coloured or ~luorescent molecules and ligands, pre~erably residues capable of binding in~unological.ly.
Examples o~ the latter are in particular haptens such as digoxigenin and also vitamins such as biotin. In the case of enzymes the detection is usually carried out via a colour-forming reaction catalyzed by the enzyme.
Coloured or fluorescent molecules can be detected directly by photometric or fluorometric means. Ligands are ~sually contacted with a corresponding enzyme-labelled or dye-labelled receptor which has affinity to the ligands and are d~tected by this means.
A detectably labelled nucleic acid hybricl i5 understood as a partially double-stranded hybrid of at least two mlcleic acids. In this case either both nucleic acids or only one of them may be detectably labelled as described above. It is important that the nucleic acid hybrid has ~ ~ r~
a sinyle-stranded region. In a so-called sandwich test the hybrid preferably consists of the (non l~belled) analyte nucleic acid and a detectably labelled detector probe.
An immobilizable nucleic acid probe is understood as a nucleic acid probe which is modified by a residue which is not presenk in the usual nucleic acidsu Su~h residues are residues which have an affinity to another material.
Examples are partners of a biospecific reaction between a ligand and a receptor for example an immunological reaction, a reaction between sugar and lectin or between vitamin and binding protein. Particularly preferred residues are haptens or biotin. In the method accorcling to the present invention the residue on the immobilizable nucleic acid probe is di~fererlt from the det~ctable residue of the detectably labelled nucleic acid.
The steps of the method of detection describPd in the following are known as indlvidual steps or can be carried out analogou~ to known m~th~ds. Reference ~g in particular made to Molecular Cloning, Editor Sambrook et al., CSH 1989. These also include the known methods for the production of labelled nucleoside triphosphates, the chemical synthesis of modified ~nd unmodified oligonucleotides, the choice of hybridization conditions by which means a specificity can be achieved which i.s dependent on the extent of homology between the nucleic acids to be hybridized, their GC content and their length, as well as the formation of nucleic acids from nucleoside triphosphates with the aid of polymerases and if desirsd, using so-called primers. An essential feature of the present invention is the use of a special capture probe.
2 ~ 3 ~43 In a first step of the method according to the present invention a detectably lahelled nucleic acid or a detectably labelled nucleic acid hybrid is produced using the analyte nucleic acid. This can for example be carried out by detectably labelling the analyte nucleic acid itself. This can be achieved by the enzymatic incorporation of labelled nucleoside triphosphates or by elongating the analyte nucleia acid with labelled nucleoside triphosphates (tailing). A furthex possibility is to amplify a region of the analyte nucleic acid whila incorporating detectably labelled mononucleoside triphosphates or detectably labelled oligonucleotides. Many methods are known for this from the state of the art.
One possibility is to carry out an amplification ~y m~an~ of the pol~mera3e chain reaction aacording to EP-A-0 200 362 using the analyte nucleic acid as the template nucleic acid. In this case two primers are used one of which is complementary to a region of the analyte nucleic acid and the other is complementary ko a part of the oppo~ite strand of the analyte nucleic acid to form a multitude of copies of the strand and opposite strand of the analyte nucleic acid by elongation of the pri.mer on the template. In order to produce the detectably lab~lled nucleicc acid, at least one detectably labelled mononucleoside triphosphate is used in the elongation of the primer or a labelled primer is used.
A further possibility ar.ises from the use of the ligase chain reaction according to W0 90/06376 if at least one of the oligonucleotides is detectably labelled.
A further possibility is described in EP-A-0 329 822. In this process a DNA strand is formed which is - 6 - ~ Q 7 ~
complementary to substantial parts of the analyte nucleic acid and a primer containing a promoter is incorporated. After degradation of the analyte nucleic acid, a transcribable double strand is ~ormed which can be used to form a multitude of detectably labelled transcripts while incorporating labelled mononucleoside triphosphates.
The method according to the present invention has proven to be particularly efficient when in the form of a sandwich nucleic acid assay e.g~ according to EP-A-O 192 168. The method according to the present invention differs from ~he ~nown sandwich assays in that a particular type of capture probe is used.
In a ~urther step the detectably labelled nucleic acid or the detectably labelled nucleic acid hybrid is brought into contact with an immobllizable nucleic acid probe under hybridization conditions.
The immobilizable nucleic acid probe according to the present invention contains two or more nucleotides modified by immobili~able groups which are not directly adjacent in the nucleotide sequence. Apart from the number of ligands (reporter groups) the distance between the ligands is crucial and should be preferably 10 or more nucleotides in order to observe this efEect. For example oligodeoxyribonucleotide probes which contained 5 biotin residues (biotin corresponds to the ligand) coupled in direct succession and were used as capture probes to bind an analyte to a streptavidin matrix did not result in an increase in the sensitivity in the total assay compared to a reference probe which was only linked to one biotin residue ~see example 2). The "capture probe" can ~e an oligonucleotide or a 7 ~ ;3~j~S
polynucleotide (DNA ~r R~) which can hybridlze in a suitable manner with the analyte nucleic acid to be detected. It can be single-stranded (e.g.
oligodeoxyribonucleotide, oligoribonucleotide) or double-stranded ~e.g. plasmid, fragment). In the latter case the capture probe has to be denatured be~ore hybridization with the analyte. A particularly preferred embodiment utilizes nucleic acid probes in which the terminal nucleotide~ of the nucleic acid probe are modified in each case.
An oligodeoxynuc}eotide is preferred which has a length between ll and 40 nucleotides whereby the label is appropriately attached to the 3'- and 5'- end in ordler to meet the requirement for distance as described above.
The immobilizable residues can in principle be bound to the base part or to the ~ugar part or to the phosphate part of the nucleotides. Such immobilizable nucleic acid probes can be produced acaording to known method~ or analogous to known methods. The attachment of a ligand to nucleotides with modi~ied bases is described for example in Nucleic Acids Res. 15 ~12), p. 4857 to 4876 ~1987); Nucleic Acids Res. 16 (9), p. 4077 to 4095 (1988); Nucleic Acids Res. 17, p. 4643 to 7650 (1989) or can be carried out by introducing a modified nucleotide which already contains the ligand (e.g. Nucleic Acids Res. 18 (15), p. 4355 to 4360 (1990). A ~urther suitable chemical method for incorporating several primary amino groups in synthetic oligonucleotides which can then be used for binding to an immobilizable residue is described in Nucleic Acids Res. 17 (18), p. 7179 to 7194 ~1989). The attachment o~ the ligand to a nucleic acid can also be carried out by a bisulfide-catalyzed transamination (Nucleic Acids Res. 12 (2), p. 989 to - 8 - ~f~
1002 (1984)). The attachment of several immobilizable xesidues can al60 be carried out by enz~matic methods e.g. by tailing using a nucleoside triphosphate containing the appropriate residue tDNA 5 (4), p. 333 to 337 (1986~) or by random priming (Analyt. Biochem~ 132, p. 6 lg~3)~
Chemically synthesized oligonucleotides are particularly preferred within the scope of the present invention since they hava the advantage that the distance between two modified nucleotides can be exactly determined bef orehand.
The use of multiply labelled nucleic acids as detection probe~ was known previously. For example in EP-A-0 330 221 an oligonucleotide or a polynucleotide is labelled with biotin and subse~uently biotin is detected by means o~ a streptavidin-enzyme complex. The separation of two ~iotin-dUMP residues in the nucleotide sequence by simultaneous incorporation of dTTP is de~cribed as being more of a disadvantage. In Nucleic Acids Res. 1~ (15), p~ 4358 it is shown that by attaching several immobilizable residues to nucleic acids their binding to solid phases can be improved.
In Nucleic Acids Res. 18, p. 4345 to 4354 (1990) multiply labelled nucleic acids are also used as a detection probe. The production of multiply labelled nucleic acids in which the label is attached to the phosphate residue .is described in W0 90/08838.
If the sample contains analyte nucleic acid, a nucleic acid hybrid forms from the detectably labelled nucleic acid or the detectably labelled nucleic acid hybrid and - 9 - l~v ~
the immobilizable nucleic acid probeO In the m~thod according to the present invention this is bound via the immobilizable residue to a solid phase. The surface of the solid phase contains groups which have an affinity to the immobilizable residue o~ the nucleic acid probe, for example a receptor. If the immobilizable group is a hapten, the solid phase preferably contclins an antibody against this hapten. In the case of biotin the solid pha~e contains a biotin binding protein such as avidin or streptavidin.
The amount of immobilizable hybrid is a measure for the amount or the presence of the analyt~ nucleic acid. This amount can be detscted via the amount of label which is immobilized on th~ solid phase. This is preferably carried out in a known manner and depends on the type of label used. Before carrying out the detection reaction, the solid phase is preferably removed from the liquid phase. This at the same time results in the removal of the starting material ~or the production o~ the detectably labelled nucleic acid or for the detectably labelled nucleic acid hybrid or of an excess detec~ably labelled nualeic acid probe in the case of a sandwich hybridization togather with the li~uid. The detection of a nucleic ac~d by the method according to the present invention, especially if the test is to be carried out quantitatively, encompasses comparing the measurement signal which was obtained with the sample of unknown analyte nucleic acid content with the measurement signal or measurement signals which were obtained with one or several samples with known analyte nucleic acid content.
The use o~ a calibration curve is preferred which can also be provided in the form of entered data.
1 0 ;~ ?~ (.J ~
The invention in addition concerns the use of a nucle.ic acid which contains two or more nucleotides modified ~y immobilizable groups which are not directly adjacent in the nucleotide sequence ~or immobilizing nucleic acids.
This aspec~ is based on the ~act that the nucleic acid probes according to the present invention guarantee a ~urprisingly improved immobilization~ T~he method can for example be used in the affinity separation of nucleic acids.
The invention also relates to a system for the detection of nucleic acids which contains one or several nucleic acids containing two or more nucleotides modified by immobilizable groups which are not directly adjacent in their respective nucleotide sequence and at least one solid phase which has a specific af f inity to the lmmobllizable groups of tha nucleic acid. Be~ore starting a detection method it is pr~ferable that the nucleic acids and the sclid phase are present separated ~rom one another in the system. ~n addition the system can also contain further components which are necessary or helpful for the detection of nucleic acids~ These in particular include pH buffers and reagents for detecting the label.
Fig. 1 shows a diagram of the binding of an analyte nucleic acid A via a capture probe F according to the present invention to a solid phase coated with streptavidin S in a sandwich test. The detectably (represented by a rhombus) labelled detector probe is denoted D.
Fig. 2 shows the binding to the wall of a labelled analyte nucleic acid A' via a capture probe according to the present invention to a solid phase coated with streptavidin S.
Fig. 3 illustrates the increase of the san~itivity of a sandwich nucleic acid test in which thQ immobilizable groups are located on adjacent nucleotide6 by separating the groups according to the present invention. It is clear that the increa~e in sensitivity when using only two biotin residue~ which are each at t:he ends of the oligonucleotide is unexpectedly even more than when 10 biotin residues are used at one end ~see also example 2).
Fig. 4 shows the increase in sensitivity in the sandwich test when using tws biotin residues compared to the use of one biotin resldue with an oligonucleotide length of 30 nucleotides.
Fig. 5 shows the comparison of sensitivity of Figure 4, but with oligonucleotides having a length o~ 20 nt.
Fig. 6 shows the compari~on of sen~itivity for a method in which an analyte nucleic acid wa5 amplified and at the same time labelled by PCR and in which nucleic acid probes with a length of 15 nucleotides and having one or two biotin residues were used as the capture probe.
12 - ~ Q 7 3 9 ~ ~
The curves shown in figures 4 to 6 can also be used as calibration curves for determining an unknown a~ount of an analyte nucleic acid.
Fig. 7 shows the formula for the aminomodifier II.
The invention is elucidated in more detail by the following example~.
- 13 ~ ,~J,~
PrQduction of oli~onucleot~des A: Explanatory notes All oligonu~leotides were produced with the aid of a DNA synthesizer 8700 from the Biosearch Company using the "phosphoramidite method" published by Caruther~ et alO Methods Enzymol. 154, 287, 1987.
The ~-cyanoethyl~nu~leoside phosphoramidites used were obtained ~rom Roth ~Karlsruhe, GFR), D
biotino~l-aminocaproic acid-N-hydroxysuccinimide eq~ter was from Boehringer Mannheim ~Mannheim, GFR), aminomodifier 2 (AM II) from Beckmann Instrumen~s GmbH ~Fullerton, CA, USA). All other reagents and solvents were used in the best ~uality available.
The purification of the biotlnylated oligonucleotide~ was carried out by HPLC (Spectra Physics, Darmstadt, GFR) using a Mono Q HR 5/5 ion-exchange column from Pharmacia (Freiburg, GFR) or a Cl~ column (LiChrosorb RP 18-5, CS-Chromatographie Service, Langerwehe, GFR). Spectra/Por lO00 membranes (Roth, Karlsruhe, GFR) were used for the dialysis.
B) Synthesis ~ad ~ri~ication of t e biotlnvlated oli~onucleotides The biotinylated oligonucleotides were all prepared in the Biosearch synthesizer on a 1 ~mol scale using the trityl-off as well as cleave-off programme. Since there was no carrier material available with aminomodifier 2, a th~midine carrier was used and subsequently aminomodifier 2 (see Fig.
7) was condensed to this according to the standard coupling program ~0.1 M in acetonitrile).
A~er the amlnomodifier, a further th~midine was coupled to the 5' end in order to protect the primary hydroxyl group of the aminomodi.~ier ~rom attack during the ammonia treatment and the glyceryl residue from being again partially cleaved o~f. A~ter completion of the .~ynthesis and cleavage of the protecting groups with concentrated NH3 solution at 55C/5h, all solvents were removed in a vacuum, the residue was taken up in water and the product was purified by ion-exchange chromatography (Mono ~ HR 5~5; A~ 0.25 mM Tris/HCl, 0.3 M NaCl; ~:
0.25 Tris/HCl, 1 M MaCl; in 60 min from 0 to 100 B, flow 1 ml/min). Subsequently the oligodeoxynucleotide was desalted by dialysis (Spectra/Por 1000, Roth). Afterwards the oligonuclotides were biotinylated by taking up 5-10 O.D.260nm of the oligodeoxynucleotides in 0.5 ml 0.05 M K2HP04/KH2P0~ buffer and adding a solution of 5 mg D-biotinoyl-aminocapxoic acid-N-hydroxysuccinimide ester in 0.5 ml DMF. The reaction mixture was incubated overnight at 37C, subsequently the solvent was removed in a vacuum, the residue was taken up in redistilled water and - 15 ~ a~
excess biotin was removed by filtration. The further purification was carried out by means of reversed phase ~PLC with a gradient o~ ~: 0.1 M
triathylammonium acetate p~I 7; 5 % acetonitrile, B:
O.1 M triethylammonium acetate pH 7; 40 %
acetonitrile. The gradient was run ~rom 20 % B to 80 % B within 40 min at a flow rate of 2 ml/min.
The dif~erence in the ret~ntion time between the twice biotinylated oligonucleotides and the corresponding aminomodi~ier-modi~ied starting oligonucleotides was between 2 and 4 minutes depending on the length of the sequence. The biotinylated oligodeoxynucleotid2s could bs obtained in 1.5 to 3.5 O~D.2~0 after renewed dialysis.
~etaction o~ ~v analyte D~A in a sandwich form taklnq into account_the site o~. modification The test is carried out in a sandwich with binding of the analyte DNA to a solid phase. A partial sequence of the hepatitis B virus DNA which is present clon~d in a plasmid is used as the analyte DNA. The analyte DNA is bound to a streptavidin solid phase by means of a biotin-labelled oligonucleotide whose sequence is complementary to a region of the HBV DNA. The detection of the bound analyte DNA is then carried out by means o~
a diyoxigenin-labelled oligonucleotide which in turn is complementary to another region of the analyte DNA. This can be recognized by anti-digoxigenin antibodies which are conjugated with horseradish peroxidase and the hybrid is subsequently detected by an enzyme-catalyzed colour-forming reaction.
A dilution series of the plasmid in a range of 4~4 ~g/ml, 2.2 ~g/ml, 1.1 ~g/ml, 0.55 ~g/ml in TE buffer ~10 mM Tris-HCl, 1 mM EDTA, pH 7.5~ is prepared. In order to denature khe double-strande~ DNA, 10 ~1 ~ M
NaOH 16 added to 90 ~1 of the plasmid solution and i~cubated for 10 min at room temperatur~. 20 ~1 of the denaturation prepaxation i~ subse~uently pipetted into a well o~ a microtitre plate ~oated with streptavidin and immediately neutralized by addition of 180 ~1 hybridization solution ~50 mM Na phosphate buffer, 0.75 M NaCl, 0.075 ~ Na citrate, 0.05 % bovine ~erum albumin, pH 5.4). This results in the following concentration~ of the plasmid in the testo 5~, 100, ~00, 400 ng/ml. The hy~ridi~ation solution in addition contains 200 ng/ml of a digoxigenin-labelled oligonucleotide (labelled once with digoxigenin at the 5' end, position 287c-24~c in the HBV genome) and of a biot~n-labelled oligon~cleotide (position 2456c-2417c in the HBV genome). The te~t is carried out with oligonucleotldes havi~g different degrees of bio~in labelling:
HBV-Oli 7-4 (1 biotin, 40mer), SEQ ID NO 1:
5'-T(bio-AMII)-CATTGAGATTCCCGAGATTGAGATCTTCTGCGACGCGGCG-3' HBV-Oli 7-5 ~5 biotin, 40mer), SEQ ID NO 2:
5'-T-(bio-AMII)5-CATTGAGATTCCCGAGATTCAGATCTTCTGCGACGCGGCG-3' HBV-Oli 7-6 (10 biotin, 40 mer), SEQ ID NO 3:
5'T-(bio-AMII)10-CATTGAGATTCCCGAGATTGAGATCTTCTGCGACGCGGCG-3' 17 ~ /7 ;~ ~3 f~ J
HBV-Oli 7-7 (2 biotin, 40mer~, SEQ ID N0 4~
5'T-(bio MII)-cATTGAGATrrcccGAGATTcAGATcTTcTGcGAcGcGGcG
(AMII-bio)-T-3' The hybridization preparation i~ incubated for 3 h at 37C in the microtitre plate while shaking. A~t~r aspirating the solution it is washed 2 x 10 min with 0.3 M NaCl, 0.03 M Na citrate, 002 % Na dodecylsulate at 370c and ~ubsequently once ~or a short time at room temperature with 0.9 % NaCl in order to remove non-bound reaction partners from the test. 20 mU/ml o~ an anti-digoxigenin antibody-horseradish peroxidase conjugate in 103 mM Tris-HCl (p~ 7.5), o.s % NaCl, 1 ~ bovin~ serum albumin is added and incubated ~or 30 min at 37C while shaking. Non-bound con~uyate is removed by briefly washing three times with 0.9 % NaCl at room temperat.ure.
The deteotion r~action is started by addition of thQ
substrate solution ABTS~ ~.9 mM, 2,2'azino.di~[3-ethylbenzthiazoline sulfonic acid (6)3-dia~m~nium salt).
The incubation is carried out ~or 30 min at 37C while shaklng. The absorbance i8 subsequently measur~d at 405 nm by means of an E~ISA reader.
The results ar~ ~hown in Fig. 3.
~amp~ 3 Detection of HBV-analYte DNA in a sandwich format takinq into account the chain_len~th and the site oE
modification A d.ilution series of the plasmid which contains HBV-specific sequences is prepared in a range from 62.5 to 250 ng/ml in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. 10 ~1 5 M NaOH is added to 90 ~1 of the plasmid solution and incuhated for 10 min at room temperature~ 20 ~1 of this denaturation preparation is pipetted into a well of a microtitre plate coated with streptavidin and 180 ~1 hybridization solution (50 mM phosphate buffer, 0.75 M
NaGl, 0.075 M Na citrate, 0.05 ~ bovine serum albumin (~SA), pH 5.4) is added. 200 ng/ml o~ the biotin-labell~d captux~ oligonucleotide and of th~ digoxigenln-labelled oligonucleotides ~pos. 405-444 in the HBV
genome, 40mer, labelled twice with digoxigenin at the 3' and 5' end) are added to the hybridization solution.
Thus the analyte DNA concentration in the test is 6.25, 12.5, 25 and 50 ng/ml.
HBV-Oli 25-3 ~pos. 2327-2356, 30mer, 1 biotin), SEQ ID NO 5:
5'-T-(bio-AMII)-CACTTCCGGAAACTACTGTTGTTAGACGAC-3' H~V-Oli 25-4 (pos. 2327-2356, 30mer, 2 biotin), SEQ ID NO 6:
5'-T-(blo-AMII~-CACTTCCGGAAACTACTGTTGTTAGACGAC-~MII-bio-~-T-3' HBV-Oli 33-1 (pos. 2332-2351, 20mer, 1 biotin).
SEQ ID NO 7:
5'-T-(bio-AMII)-CCGG~AACTACTGTTGTTAG-3' HBV-Oli 33-2 (pos. 2332-2351, 20mer, 2 biotin), SEQ ID NO 8:
5'-T-(bio-AMII)-CCGGAAACTACTGTTGTrrAG-(AMII-Bio)-T-3' ~he stated positions relate to the HBV genome.
- 19 - ~?~ ~ 3 ~
The hybridi.zation preparation is shaken for 3 h at 370C.
Subsequently it is washed 2 x lo min with 0.3 M NaCl, 0.03 M Na citrate, o.~ % Na dodecylsulfate at 37OC and then once briefly wi~h o.s % NaCl at room temperature.
Su~sequently 20~ ~l of the anti-digoxigenin antibody-horseradish peroxidase conjugate (200 mU/ml in 100 mM
Tris-HCl, pH 7.5 9 0 . 9 % NaCl, 1 % BSA) is added and incubated for 30 min at 370C whil2 shaking. After washing again three times with O . 9 % NaCl, the substrate solution ~1.9 mM ABTSR) is pipetted shaken again for 30 min at 370C and th~n the absorbance is measured at 405 nm by mean6 of an ELISA reader.
The result~ ~or the 30 nt oligonucleotid~s are shown in Fig. 4, those for the ~o nt oligonucleotides are shown in Fig. 5.
B~mpla Detection of_~Ç~ Products wit~ nqlY=_and_~ e-lahelled oli~onucleotides For the DNA test for the hepatitis B viruses, the viral DNA is firstly amplified by means of the polymerase chain reaction (EP~A-0 200 362). During this amplification digoxigenin is incorporated into the DNA
in the form of Dig~ dUTP (EP-A-32~474). Subsequently this hapten allows a sensitive detection of the PCR
product in a heterogeneous immunoassay. The binding of the digoxigenin-labelled DNA to the walls of the streptavidin-coated tubes is carried out by means of a biotin-labelled oligonucleotide who~e region is complementary to a region of the PCR product. The test is again carried out using an anti-digoxigenin antibody-~ ~ ~ 3 ~ . ~
~ 20 ~
horseradi~h peroxidase conjugate and a sub~e~uent colourreaction.
HBe-positive human plasma with a virus titre of 1 x 101 hepatitis B viruses per ml is diluted in normal serum so that the virus content in the dilutions is 1 x 107 viruses/ml. For the lysi~, 10 ~l 0.2 M NaOH is added to 10 ~l of the virus dilution and incubatisd for 1 h at 37C. The lysis preparation i6 neutrali2ed by addition of 30 ~l neutrali~ation mixture (100 mM KCl, 50 mM Tris-HCl, pH 6.5, 3 mM MgCl2). 20 ~l of this solution is used in the subsequent amplification reaction (amplification conditions: 200 nM of each of the PCR prim~rs, 200 ~M
each of dATP, drTp/ dGTP, 175 ~M dTTP, 25 ~M
digoxigenin~ 2'-dUTP, 2.5 U Thermus aquQticus DNA
polymerase in 50 mM KCl, 10 mM Tris-HCl, pH 8.9, 1.5 mM
MgCl2, 0.01 % gelatin; total volume 100 ~l). The preparation is covered with a layer of 100 ~l mineral oil and incubated ~or 30 cycles in a thermo-cycler (Perkin-Elm~r):
30 sec 92C, 30 sec 50C, 60 sec 70C.
Due to the position of the PCR primers (PCR primer 1:
position 1937-1~60; PCR primar 2: position 2434c-2460c in the HBV genome) a 500 bp long DNA fragment is produced which is subsequently detected in a two-component test system in streptavidin-coated microtitre plates.
The PCR preparation is diluted in H20 in such a way that in relation to the original virus concentration of 1 x 107 viruses per ml serum, dilutions ar~ made which correspond to a range of 1 x 105 to 1 x 104 viruses/ml.
20 ~1 of the diluted PCR preparatlon is incubated in a ~inal concentration of 0.1 N NaO~ in a ~olume of 50 ~1 for 10 min at room temperature for the denaturation.
20 ~1 of the denaturation solution are pipetted together with 180 ~1 hybridization solution (1 M NaCl, 0.~ M Na citrate, 67.5 mM Na phosphate, 0.05 % BSA, p~ 6.7) into a ~treptavidin-coated well of a microtitre plate. The biotin-labelled oligonuoleotides are added at a concentration of 100 ng/ml to the hybridization buffer:
HBV-Oli 34-1 (pos. 2299-2313, 15mer, 1 biotin) r SEQ ID NO 9:
5'-T-(~MII-bio)-AGACCACCAAATGCc~3' ~BV-Oli 34-2 (pos. ~299-2313, 15mer, 2 biotin) SEQ ID NO 10~
5' T-(AMII-bio)-AGACCACCAAATGCC-(bio-AMIIH)-T-3' The hybridization preparation i5 incubated for 3 h at 37C while skaking. Subsequently the ~olution is aspirated and the well is washed three time~ with 0.9 %
NaCl. 200 ~1 of a solution of 200 mU/ml anti-digoxigenin antibody-hor~eradlsh peroxidase con~ugate in 100 mM
Tri~-HCl, pH 7.5, 0.9 % NaCl, 1 % BSA is added and incubated for 30 min at 37C. After washing again with 0.9 % NaCl the substrate reaction is started by addition of 200 ~1 ABTSR. The photometric measurement is carried out after 30 min at 405 nm.
The results are shown in E'ig. 6.
~o~uerlce Protocol SEO ID ~0 1 ~ength of sequence: 41 bases Type of sequence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position- HBV genome 2456c-2417c (Hpbadw-data bank~
Antisense Modification N: T on this is bound to aminomodifier II via 3'-0 biotin 5'-N CAT TGA GAT TCC CGA GA~ TGA GAT CTT CTG CGA CGC GGC
G-3' SEQ I~ NO_2 Length of sequence: 41 base~
Type o~ sequence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2~56c-2417c Antisense Modification N: T on this is bound to 5 aminomodifiers II via 3'-0 5 biotin 5'-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
G-3' 2 ~
Length of sequence: ~1 bases Typ~ of saquence: nucleotide se~uence Type of strand: single strand Topology: linear Type of moleculeo part of genome DNA with modifi~ations N
Position: HBV genome 2456r-2417c Antise~se Modification N: T on this is bound to 10 aminomodifiers II via 3'-0 10 biotin 5l-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
G~3' Length of se~uence- 42 base~
Type of sequence: nucleotide sequence Type of st~and: single strand Topology: linear T~pe of molecule: part of genome DNA with modifications N
Position: HBV genome 2456c-2417c Antisense Modification N at T on this is bound to the 5' end: aminomodi~ier II via 3l_0 biotin Modification N at T on this is bound to the 3' end: aminomodifier II via 5' 0 biotin 5'-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
N-3' - 2~ -. f SE QID NO_5 Length of sequence: 31 basQs Type of se~uence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2327-2356 Modification N: T on this is bound to aminomodifier II via 3'-0 biotin 5'-N CAC TTC CGG AAA CTA CTG TTG TTA ~AC GAC 3' SE ~ID NO 6 Length o~ ~equence: 32 bases Type of sequence: nucleotide sequence Type of strand: single ~trand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2327-2356 Modi~ication N at T on this is bound to the 5~end: aminomodifier II via 3'-0 biotin Modification N at T on this i.s bound to the 3'end aminomodifier II via 5'-0 biotin 5'-N CAC TTC CGG AAA CTA CTG TTG TTA GAC GAC N-3' Len~th of sequence: 2 O bases Type of sequenceO nucleotide sequence Type of strand- ~;ingle strand Topology: linear Type of molecule: part Q~ genome DNA with modi~ications N
Position: H~Y genoma 2332-2351 Modi~ication N: T on this i~3 bound to aminomodif ier II via 3 ' -O biotin 5 '-NC CG(; A~A CTA CTG TTG TTA G-3 1 ~ ;.3 Length o~ sequence: 21 basPS
Type of sequence: nucleotide sequence Type o~ ~trand: single strand Topology: linear YPQ of molecule: part of genome DNA with modi~ication~ N
Position~ HBV genome 2332-2351 Modification N at T on this is bound to the 5' end: aminomodifier II via 3'-0 biotin Modification N at T cn this is bound to the 3' end: aminomodifier II via 5'-0 biotin 5'-NC CGG AAA CTA CTG ~TG TTA GN-3 SEQ ID NO ~
Length o~ sequence: 16 bases Type of sequence: nucleotide sequence Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modifications N
Position: HBV genome 2299-2313 Modification N: T on this is bound to aminomodifier II via 3'-0 biotin 5'-N AGA CCA CCA AAT GCC-3' SE~ ID NO 10 !.~
Length of sequence: 17 bases Type of sequence: nucleotide sequenc~
Type of strand: single strand Topology: linear Type of molecule: part of genome DNA with modi~ication6 N
Position: HBV genome 2299-2313 Modification N at T on this is bound to the 5' end aminomodifier II via 3'-0 biotin Modification N at T on this is bound to the 3' end: aminomodifier II via 5'-0 biotin 5'-N AGA CCA CCA AAT GCC N-3'
Claims (16)
1. Method for the detection of an analyte nucleic acid comprising the following steps:
- hybridizing a detectably-labelled nucleic acid or a detectably-labelled nucleic acid hybrid with an immobilizable nucleic acid probe;
- immobilizing the hybrid which forms and - detecting the amount of immobilized hybrid by means of the amount of label, wherein the immobilizable nucleic acid probe contains two or more nucleotides modified by immobilizable groups which are not directly adjacent in the nucleotide sequence.
- hybridizing a detectably-labelled nucleic acid or a detectably-labelled nucleic acid hybrid with an immobilizable nucleic acid probe;
- immobilizing the hybrid which forms and - detecting the amount of immobilized hybrid by means of the amount of label, wherein the immobilizable nucleic acid probe contains two or more nucleotides modified by immobilizable groups which are not directly adjacent in the nucleotide sequence.
2. Method as claimed in claim 1, wherein the distance between the modified nucleotides is at least 10 nucleotides.
3. Method as claimed in claim 1, wherein the distance between the modified nucleotides is between 11 and 40 nucleotides.
4. Method as claimed in claim 1, 2 or 3, wherein the modified nucleotides represent the terminal nucleotides of the nucleic acid probe.
5. Method as claimed in claim 1, 2 or 3, wherein the immobilizable residues are bound to phosphate residues of the nucleotides.
6. Method as claimed in claim 4, wherein the immobilizable residues are bound to phosphate residues of the nucleotides.
7. Method as claimed in claim 1, 2 or 3, wherein the immobilizable groups are bound to the base part of the nucleotides.
8. Method as claimed in claim 4, wherein the immobilizable groups are bound to the base part of the nucleotides.
9. Method as claimed in claim 1, 2 or 3 wherein the detectably-labelled nucleic acid is an amplification product of a region of the analyte nucleic acid.
10. Method as claimed in claim 4, wherein the detectably-labelled nucleic acid is an amplification product of a region of the analyte nucleic acid.
11. Method as claimed in claim 1, 2, 3, 6 or 8, wherein the detectably-labelled hybrid is a hybrid of the analyte nucleic acid and a detectably-labelled nucleic acid probe.
12. Method as claimed in claim 4, wherein the detectably-labelled hybrid is a hybrid of the analyte nucleic acid and a detectably-labelled nucleic acid probe.
13. Method as claimed in claim 5, wherein the detectably-labelled hybrid is a hybrid of the analyte nucleic acid and a detectably-labelled nucleic acid probe.
14. Method as claimed in claim 7, wherein the detectably-labelled hybrid is a hybrid of the analyte nucleic acid and a detectably-labelled nucleic acid probe.
15. Use of a nucleic acid containing two or more nucleotides modified by immobilizable groups which are not directly adjacent in the nucleotide sequence for immobilizing nucleic acids.
16. System for the detection of nucleic acids containing - one or more nucleic acids which contain two or more nucleotides modified by immobilizable groups which are not directly adjacent in the respective nucleotide sequence and - at least one solid phase which has a specific affinity for the immobilizable groups of the nucleic acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4123540.1 | 1991-07-16 | ||
| DE4123540A DE4123540A1 (en) | 1991-07-16 | 1991-07-16 | IMMOBILIZATION OF NUCLEIC ACIDS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2073989A1 true CA2073989A1 (en) | 1993-01-17 |
Family
ID=6436267
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002073989A Abandoned CA2073989A1 (en) | 1991-07-16 | 1992-07-16 | Immobilization of nucleic acids |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0523557B1 (en) |
| JP (1) | JP2644419B2 (en) |
| KR (1) | KR960004039B1 (en) |
| AT (1) | ATE132538T1 (en) |
| AU (1) | AU640089B2 (en) |
| CA (1) | CA2073989A1 (en) |
| DE (2) | DE4123540A1 (en) |
| DK (1) | DK0523557T3 (en) |
| ES (1) | ES2083629T3 (en) |
| FI (1) | FI923239A (en) |
| IE (1) | IE922298A1 (en) |
| IL (1) | IL102487A0 (en) |
| NO (1) | NO922794L (en) |
| NZ (1) | NZ243525A (en) |
| ZA (1) | ZA925259B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4239531A1 (en) * | 1992-11-25 | 1994-05-26 | Gabor Dr Igloi | Reagent for coupling various substances to nucleic acids and process for its production |
| CA2119701A1 (en) * | 1993-04-16 | 1994-10-17 | Colleen M. Nycz | Nucleic acid assay procedure |
| US6821727B1 (en) | 1993-11-15 | 2004-11-23 | Applera Corporation | Hybridization assay using self-quenching fluorescence probe |
| US5538848A (en) * | 1994-11-16 | 1996-07-23 | Applied Biosystems Division, Perkin-Elmer Corp. | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
| DE19516196A1 (en) * | 1995-05-08 | 1996-11-14 | Boehringer Mannheim Gmbh | Method for the quantitative detection of nucleic acids |
| DE19525632C2 (en) * | 1995-07-14 | 1997-07-17 | Bag Biolog Analysensystem Gmbh | Sequence-specific nucleic acid detection method and reagent system for its implementation |
| DE19533354C2 (en) * | 1995-09-08 | 1999-07-01 | Derya Dr Ozkan | Methods and their applications for sequence-specific detection and quantification of nucleic acids |
| DE19537952A1 (en) * | 1995-10-12 | 1997-04-17 | Boehringer Mannheim Gmbh | Method for the detection of an analyte |
| DE19548680A1 (en) * | 1995-12-23 | 1997-06-26 | Boehringer Mannheim Gmbh | Method for the quantitative detection of specific nucleic acid sequences |
| US5840879A (en) * | 1996-12-06 | 1998-11-24 | Wang; Edge R. | Reagents and solid supports for improved synthesis and labeling of polynucleotides |
| ES2129001B1 (en) * | 1997-07-18 | 2000-04-16 | Pharmagen S A | METHOD FOR THE DETECTION AND VISUALIZATION OF A SPECIFIC DNA SEQUENCE. |
| DE19835856A1 (en) * | 1998-08-07 | 2000-02-17 | W Kurt Roth | Oligonucleotide primers and probes, for the detection of hepatitis B virus, are used to amplify, by polymerase chain reaction, a section of the hepatitis B virus genome |
| DE102022204638A1 (en) * | 2022-05-12 | 2023-11-16 | Robert Bosch Gesellschaft mit beschränkter Haftung | Method and microfluidic device for denaturing a nucleic acid |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4582789A (en) * | 1984-03-21 | 1986-04-15 | Cetus Corporation | Process for labeling nucleic acids using psoralen derivatives |
| US4868105A (en) * | 1985-12-11 | 1989-09-19 | Chiron Corporation | Solution phase nucleic acid sandwich assay |
| JPS638396A (en) * | 1986-06-30 | 1988-01-14 | Wakunaga Pharmaceut Co Ltd | Poly-labeled oligonucleotide derivative |
| JPS6455198A (en) * | 1987-06-11 | 1989-03-02 | Nippon D P C Corp | Method for hybridization of nucleic acid |
| US5082830A (en) * | 1988-02-26 | 1992-01-21 | Enzo Biochem, Inc. | End labeled nucleotide probe |
| US5185243A (en) * | 1988-08-25 | 1993-02-09 | Syntex (U.S.A.) Inc. | Method for detection of specific nucleic acid sequences |
-
1991
- 1991-07-16 DE DE4123540A patent/DE4123540A1/en not_active Withdrawn
-
1992
- 1992-01-01 ZA ZA925259A patent/ZA925259B/en unknown
- 1992-07-09 AU AU19534/92A patent/AU640089B2/en not_active Ceased
- 1992-07-10 EP EP92111743A patent/EP0523557B1/en not_active Expired - Lifetime
- 1992-07-10 DE DE59204886T patent/DE59204886D1/en not_active Expired - Lifetime
- 1992-07-10 AT AT92111743T patent/ATE132538T1/en not_active IP Right Cessation
- 1992-07-10 ES ES92111743T patent/ES2083629T3/en not_active Expired - Lifetime
- 1992-07-10 DK DK92111743.8T patent/DK0523557T3/en active
- 1992-07-13 NZ NZ243525A patent/NZ243525A/en unknown
- 1992-07-13 IL IL102487A patent/IL102487A0/en unknown
- 1992-07-15 NO NO92922794A patent/NO922794L/en unknown
- 1992-07-15 FI FI923239A patent/FI923239A/en not_active Application Discontinuation
- 1992-07-15 IE IE229892A patent/IE922298A1/en not_active Application Discontinuation
- 1992-07-16 JP JP4189189A patent/JP2644419B2/en not_active Expired - Lifetime
- 1992-07-16 KR KR1019920012681A patent/KR960004039B1/en active IP Right Grant
- 1992-07-16 CA CA002073989A patent/CA2073989A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU1953492A (en) | 1993-04-29 |
| FI923239A0 (en) | 1992-07-15 |
| JPH05199899A (en) | 1993-08-10 |
| DE4123540A1 (en) | 1993-01-21 |
| DK0523557T3 (en) | 1996-05-06 |
| IE922298A1 (en) | 1993-01-27 |
| KR930002371A (en) | 1993-02-23 |
| EP0523557A1 (en) | 1993-01-20 |
| KR960004039B1 (en) | 1996-03-25 |
| NZ243525A (en) | 1993-11-25 |
| AU640089B2 (en) | 1993-08-12 |
| ES2083629T3 (en) | 1996-04-16 |
| FI923239A (en) | 1993-01-17 |
| NO922794L (en) | 1993-01-18 |
| JP2644419B2 (en) | 1997-08-25 |
| IL102487A0 (en) | 1993-01-14 |
| NO922794D0 (en) | 1992-07-15 |
| ATE132538T1 (en) | 1996-01-15 |
| DE59204886D1 (en) | 1996-02-15 |
| ZA925259B (en) | 1994-01-17 |
| EP0523557B1 (en) | 1996-01-03 |
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| FZDE | Discontinued |