Classifications

• • 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
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• the invention particularly relates to a method for the sequence-specific detection of RNA or DNA molecules (hereinafter referred to as target nucleic acids) by means of solid-phase-bound labeled oligonucleotides, which are referred to below as probe nucleic acids.
• target nucleic acids RNA or DNA molecules
• probe nucleic acids solid-phase-bound labeled oligonucleotides
• probe nucleic acids which can be DNA or DNA / PNA chimeras, are able to form intramolecular secondary structures which contain double-stranded regions due to complementary regions in the base sequence.
• These double-stranded areas contain sequence motifs which can be recognized and cleaved by double-stray-specific nucleases (restriction donors) and are referred to here as cleavage modules.
• the formation of the intramolecular cleavage modules within the probe nucleic acids is prevented by hybridization of the probe nucleic acids with complementary target nucleic acids from the sample to be examined, so that after the digestion with double-strand-specific nucleases, the hybridized probe nucleic acids from the non-hybridized probe Nucleases can be distinguished.
• the method comprises the specific detection of different target nucleic acids, the base sequence of which is not identical, by differently labeled probe nucleic acids, all of which are immobilized on a point or a defined area.
• the miniaturized arrangement of many such nucleic acids in the smallest area is well known from biochip technology.
• an unlabeled probe nucleic acid is synthesized by in situ oligonucleotide synthesis [Fodor, S.P.A. et al., "Very large scale immobilized polymer synthesis” U.S. Pat. No. 5,424,186], or printing process [Cheung, V.G. et al., “Making and reading microarrays,” Nature Genetics, vol. January 21, 1999; Bowtell, D.D.L, "Options available - from start to finish - for obtaining expression data by microarray", Nature Genetics, vol. 21, 1999] placed on a solid matrix and covalently linked to it.
• the probe nucleic acids are organized in the form of so-called “spots" on the surface of the DNA array. It is not possible to determine the amount of nucleic acids immobilized on the solid matrix before a hybridization experiment. Only by hybridizing the DNA array with the labeled sample nucleic acid and a second, labeled sample nucleic acid (double labeling, Wang, B., "Quantitative microarray hybridization assays", US Pat. No. 6,004,755), which serves as an internal standard , it is possible to computationally compensate for differences in the amount of the unlabeled probe nucleic acids immobilized on the solid matrix.
• Another method uses the principle of the nuclease protection test [Sambrook, J. et al., "Molecular Cloning” 2001, 3rd Edition, Cold Spring Harbor Laboratory] in order to use non-hybridized, ie single-stranded, labeled probe nucleic acids degrading single strand specific nucleases [Kumar, R. et al., "Nuclease protection assays", US Pat. 5,770,370]. The accuracy of this method depends above all on the stability of the duplexes consisting of probe and target nucleic acids and the specificity of the single-strand-specific nucleases used.
• nucleic acid duplex which can be a DNA / DNA, DNA / RNA, DNA / PNA, RNA / RNA, RNA / PNA or PNA / PNA duplex, is determined by the number and strength of the Watson Crick specified base pairings between the complementary strands mediated by hydrogen bonds [Lewin, B., "Genes VI", 1997, Oxford University Press (and other popular textbooks in molecular biology)].
• nucleic acid duplexes are influenced by the surrounding medium, H 2 O, exposed, which weakens the hydrogen bonds between the complementary strands.
• the end regions of the duplexes are partly single-stranded and can be cleaved by the nucleases under conditions which promote the reaction of single-strand-specific nucleases (30-37 ° C.).
• Another disadvantage of this method is that single-strand-specific nucleases such as S1 nuclease, mung bean nuclease, RNase A, RNase T1, exonuclease VII, Bai 31 nuclease, micrococcus nuclease or nuclease P1 do not cleave nucleic acids in a sequence-specific manner and break down double-stranded regions very easily if that The quantitative ratio of nucleic acid and nuclease in the reaction mixture is not exactly titrated [Sambrook, J. et al., "Molecular Cloning" 2001, 3rd Edition, Cold Spring Harbor Laboratory].
• Molecular beacons are probe nucleic acids that can form intramolecular secondary structures and whose ends are covalently linked to different fluorophores that are brought into close spatial proximity to one another as a result of the intramolecular secondary structure.
• One of the two fluorophores quenchers
• absorbs the photons emitted by the other fluorophores emitters.
• the hybridization of a molecular beacon with a target nucleic acid dissolves the intramolecular secondary structure and the light emitted by the excited fluorophore (emitter) can no longer be absorbed by the quencher.
• DNA arrays can currently immobilize more than 10,000 "spots" per cm 2 , ie more than 10,000 different nucleic acid probes [Bowtell, DDL, "Options available - from start to finish - for obtaining expression data by microarray", Nature Genetics , vol. Jan 21, 1999].
• the maximum number of distinguishable sample points (“spots”) is determined by the smallest technically achievable point size. Since liquid quantities are transferred on a nanoliter scale during array production, this depends on physical quantities such as viscosity and surface tension of the transferred liquids.
• sample points Another parameter that limits the size of the sample points is the optical resolution of the light microscope, since all devices for detecting fluorescence, luminescence or phosphorescence work with optical systems that correspond to those of a light microscope (Confocal Laser Scanning Microscope). It is therefore not possible to fix any number of sample points ("spots") on a DNA array.
• the present invention therefore relates to a method for the detection of target nucleic acid by hybridization, wherein in the method
• a) hybridization is carried out with at least one probe nucleic acid which is bound at one end to a solid phase.
• Each probe nucleic acid has a target nucleic acid sequence which is flanked at the 3 'end by a short nucleic acid sequence and at the 5' end by a complementary nucleic acid sequence which can form a short DNA double strand. This can be cleaved by a double-strand-specific nuclease (restriction endonuclease) and thus represents a cleavage module.
• the probe nucleic acid has a label at the other end, which is not bound to the solid phase; b) at least one treatment with at least one double-strand-specific nuclease is carried out.
• Digestion with the nuclease only cuts those cleavage modules that have formed a double strand. If no double strand was able to form because the target nucleic acid hybridized with a nucleic acid to be detected, no double-stranded cleavage module is formed and the nucleic acid is not cut.
• probe nucleic acids which contain different target sequences are used in one method approach.
• probe nucleic acids are used in one method, which have different cleavage modules that can be cleaved again by different double-strand-specific nucleases.
• the probe nucleic acids can also have different labels, the labels being fluorophores and / or parts of a binding pair.
• the probe nucleic acids used according to the invention contain different variables.
• the target sequences can be varied and, as a result, very different nucleic acid sequences can be detected in the sample to be examined.
• the cleavage modules can contain recognition sequences for various restriction endonucleases.
• the probe nucleic acids can be digested either in parallel or sequentially with different restriction endonucleases.
• the probe nucleic acids can also have different labels.
• the corresponding DNA array can then be configured and several different treatment and evaluation steps can be carried out either in parallel or sequentially, and a maximum of information can be obtained in this way.
• the present invention also relates to a kit for handling hybridizations with target nucleic acids, it being possible to use the kit to carry out at least one hybridization with at least one probe nucleic acid used according to the invention.
• sequence-specific detection of target nucleic acids takes place in methods such as Northern blots, Southern blots or nuclease protection assays by detecting the formation of hybrids (duplexes) from target nucleic acids and labeled probe nucleic acids, the DNA, RNA or can be PNA.
• the unlabeled probe nucleic acid is bound to a solid matrix and is hybridized with labeled cDNA or cRNA (hereinafter referred to as sample nucleic acids).
• hybridization events in nucleic acids are detected by the detection of fluorescence, chemiluminescence, chemifluorescence or radioactivity [Sambrook, J. et al., "Molecular Cloning” 2001, 3rd Edition, Cold Spring Harbor Laboratory].
• fluorophores such as fluorescein, lissamin, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) can be used for fluorescent labeling of sample or probe nucleic acids. can be used [Kricka, L: “Non isotopic DNA probe techniques", 1992, Academic Press, San Diego].
• fluorophores in addition to the fluorophores listed here, other fluorophores not mentioned here can also be used to label nucleic acids. These include all fluorophores that can be covalently linked to nucleic acids and whose excitation and emission maxima are in the infrared range, in the visible range or in the UV range of the spectrum. If sample or probe nucleic acid is labeled with parts of a binding pair such as biotin, digoxigenin or other haptens, after hybridization the second part of the binding pair conjugated with a detectable label (streptavidin or anti-digoxigenin AK) is incubated with the duplexes.
• a binding pair such as biotin, digoxigenin or other haptens
• the detectable label of the second part of the binding pair can be a fluorophor or an enzyme (alkaline phosphatase, horseradish peroxidase, etc.) which converts a substrate under light emission (chemiluminescence or chemifluorescence) ["Fluorescent and Luminescent Probes for biological activity", 1999, 2nd Edition , Mason, WT ed.].
• the non-radioactive labeling of nucleic acids takes place through the enzyme-catalyzed synthesis of DNA or RNA in the presence of nucleotide triphosphates, the nucleotide bases of which are covalently linked with fluorophores, parts of a binding pair (e.g.
• biotin, digoxigenin or other haptens or reactive groups (NH 2 or SH) are linked.
• DNA or RNA polymerases AMV reverse transcriptase, MMuLV reverse transcriptase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, Taq polymerase, Klenow fragment , DNA polymerase and others is catalyzed, these modified nucleotide triphosphates are incorporated into the newly emerging nucleic acid [Sambrook, J. et al., "Molecular Cloning” 2001, 3rd Edition, Cold Spring Harbor Laboratory].
• the integrity of the mRNA, which is used as a template for the synthesis of the labeled sample nucleic acids is of crucial importance.
• RNA population is degraded by RNases
• the sample nucleic acid synthesized from this RNA population is, on the one hand, only very weakly labeled and, on the other hand, not representative of the transcription state prevailing in a cell type, tissue or organism to be examined.
• a representative sample nucleic acid can only be synthesized from absolutely intact mRNA.
• Nucleotide triphosphates the bases of which have been modified with the groups listed above, are incorporated by all known DNA or RNA polymerases which catalyze the synthesis of DNA or RNA with a considerably lower efficiency than unmodified nucleotide triphosphates [Molecular Dynamics Inc., "Fluorescent DNA Labeling by PCR", 1999, Molecular Dynamics Application Note # 62].
• the yield of labeled probe nucleic acid is low compared to radioactive systems and the loss of sample material due to the purification steps following the synthesis is very high.
• sample material such as culture cells or tissue samples, from which the mRNA to be analyzed is isolated
• sample material such as culture cells or tissue samples
• mRNA to be analyzed is isolated
• DNA arrays in areas such as. B. clinical diagnostics, in which the amount of available for analysis
• nucleic acids have the property of attaching to the surface of dirt particles. For this reason, the detection of solid-phase-bound, unlabeled nucleic acids by hybridization with labeled nucleic acids can be disturbed by dirt particles, especially on DNA arrays false positive results
• the method according to the invention can be used for expression analyzes in which the sequence-specific hybridization of unlabeled target nucleic acid with solid-phase-bound probes can be determined quantitatively irrespective of the integrity of the sample nucleic acids, while the maximum number of different probe nucleic acids detectable on a surface can be determined by physical limits In the field of optics and "microfluidics" is limited, a DNA array according to the invention is disclosed with a system which, not limited by the physical limits in the field of optics and "microfluidics", the quantity compared to systems corresponding to the prior art the probe nucleic acids detectable on a defined area can be significantly increased
• a method for the handling of hybridization events on solid-phase-bound probe nucleic acids which, by using solid-phase-bound labeled probes, standardizes the measurement results, the use of unlabeled sample or target nucleic acids and enables simultaneous analysis of the expression of different target nucleic acids at a defined point.
• the intramolecular secondary structure and thus the double-stranded region are dissolved and can no longer be recognized and cleaved by double-strand-specific endonucleases.
• Non-hybridized, solid-phase bound probe nucleotides are cleaved enzymatically.
• the labeled part of the probe nucleic acids according to the invention is separated from the surface by the enzymatic cleavage and diffuses into the surrounding medium and can optionally be washed out.
• the fluorescence of the hybridized, non-enzymatically cut probe nucleic acids is measured.
• the signal-background ratio of the method depends exclusively on the quality of the double-strand-specific endonucleases used and the completeness of the separation of the non-hybridized probe nucleic acids and corresponds to that of hybridization with radioactively labeled nucleic acids.
• probe nucleic acids of different sequence specificity as there are fluorophores can be immobilized on a defined area or point on a surface (spot) who are concerned about their Have the excitation and emission maxima spectrally differentiated.
• the sensitivity of the present method can be increased as desired by the number of fluorophores covalently linked to the probe nucleic acid.
• parts of a binding pair such as digoxigenin, biotin or other haptens can also be used to label the probes bound to the solid phase.
• the molecules that specifically bind the various haptens are covalently linked to enzymes with different substrate specificities. These enzymes can be alkaline phosphatase, peroxidase, acid phosphatase and others.
• enzymes can be alkaline phosphatase, peroxidase, acid phosphatase and others.
• any number of probe nucleic acids of different sequence specificity can be immobilized on a defined area or point on a surface (spot).
• Target nucleic acids can be unlabeled DNA, cDNA, cRNA or mRNA.
• target or Sample nucleic acid is used, which is complementary to the detection module of the probe nucleic acid, since the probe nucleic acid is marked in the present system.
• Another advantage of the system described is that the sensitivity of the detection is not dependent on the labeling efficiency of the sample nucleic acids, but only on the labeling of the probe nucleic acid. But this can be determined much more precisely.
• a probe nucleic acid according to the invention is shown schematically in FIG. 1A.
• a typical probe nucleic acid has the following components:
• At least one functional group (1) such as an amino group (NH2), thiol group (SH), or part of a binding pair, e.g. Biotin, digoxigenin for binding to a solid phase.
• NH2 amino group
• SH thiol group
• a binding pair e.g. Biotin, digoxigenin for binding to a solid phase.
• a sequence section ⁇ (recognition and cleavage module), which has a length of preferably 5 to 12 nucleotides, consists of DNA and the 3 'end of which is covalently connected to the end of the spacer module which is connected to the solid phase. Sequence section ⁇ contains the recognition sequence for a restriction endonuclease.
• a sequence section ⁇ (detection module) which has a length of preferably 12 to 30 nucleotides, the 3 'end of which is covalently linked to the 5' end of sequence section ⁇ (recognition and cleavage module) and can be DNA, RNA or PNA
• Sequence section ⁇ represents the portion of the molecule which, as a probe with a target nucleic acid to be detected, can form a heteroduplex under suitable reaction conditions.
• the nucleotides and / or the sugar-phosphate or pseudopeptide backbone of sequence section ⁇ can be covalently linked to fluorophores.
• a sequence section ⁇ ' (recognition and cleavage module), which consists of DNA and whose 3' end is covalent with the 5 ! - End of sequence section ß is linked.
• the sequence of section ⁇ ' corresponds to the reverse complementary sequence of sequence section ⁇ .
• a branching module [Newcome, GR et al .: “Dendritic Molecules: Concepts, Synthesis, Perspectives", 1996, VCH Publishers] (not shown) which is covalently connected to the end of the spacer module (3), which is not connected to is connected to the 5 'end of sequence section ⁇ '.
• n further branch modules can be bound to the individual ends of this branching module, so that there is a maximum number of 3 ⁇ ends, with each end
• the probe is bound to a solid matrix via element (1).
• This solid surface can be, among other things, a flat surface, which can also be convex or concave, a fiber or a micro- or nanoparticle made of inorganic or organic material.
• the inventive, bound to such a solid matrix Probe nucleic acids are referred to below as a DNA array.
• Sequence sections ⁇ and ⁇ ' are complementary to one another and, under suitable conditions, can form a double-stranded region, the duplex ⁇ - ⁇ ' (see FIG. 1B).
• the formation of a hairpin structure by intramolecular duplex formation is thermodynamically favored for this molecule compared to the single-stranded conformation.
• the molecule is present exclusively as a hairpin structure with intramolecular duplex ⁇ - ⁇ ', which can be cleaved in a sequence-specific manner by restriction endonucleases.
• the elements (1), (2) and possibly a few nucleotides of the element ( ⁇ ) remain bound to the solid phase. Labeled elements of the probe diffuse into the surrounding medium and are optionally removed by washing.
• the probe By hybridization with a target nucleic acid, which can be RNA or DNA and whose sequence is complementary to the sequence of sequence section ⁇ , the probe is partially present as a duplex with the sample nucleic acid.
• a target nucleic acid which can be RNA or DNA and whose sequence is complementary to the sequence of sequence section ⁇
• the sequence segments ⁇ and ⁇ ' are single-stranded and cannot be cleaved by restriction endonucleases or other double-strand-specific nucleases.
• the equilibrium melting temperature T m ( ⁇ ) of sequence section ⁇ must be higher than that of sequence section ⁇ or ⁇ '; thus T m (ß)> T m ( ⁇ ) [Bonnet, G.
• the equilibrium melting temperature T m ( ⁇ ) of the heteroduplex formed from sample nucleic acid and sequence section ⁇ is between 10'C and 25 "C. higher than the equilibrium melting temperature T m ( ⁇ ) of the intramolecular duplex ⁇ - ⁇ '.
• a probe - Nucleic acid which can form a secondary structure, has an increased sequence specificity compared to a linear probe nucleic acid with the same target nucleic acid.
• the difference in the equilibrium melting temperature ⁇ T "compared to a probe / target nucleic acid duplex, which has no base mismatch a probe / target nucleic acid duplex that has a base mismatch is with probe nucleic acids, which can form a secondary structure, about twice as high as with linear probe nucleic acids [Bonnet, G. et al., "Thermodynamic basis of the enhanced specificity of structured DNA probes", 1999, Proc.Natl.Acad.Sci., vol , 96].
• the method according to the invention can preferably be used for multiplex analyzes.
• n different probe nucleic acids which differ from one another in sequence section ⁇ , in the excitation and emission spectrum of the fluorophores [Vet, J.A.M. et al., "Multiplex detection of four pathogenic retroviruses using molecular beacons", 1999, Proc.Natl.Acad.Sci., vol. 96; Marras, S.A.E. et al., “Multiplex detection of single-nucleotide variations using molecular beacons", 1999, Genetic Analysis; Biomolecular Engineering, vol.
• the hybridization of n different target nucleic acids of different sequences with n different probe nucleic acids can be analyzed simultaneously in this way. If the sequence sections ⁇ and ⁇ 'of the probe nucleic acids contain n different recognition sequences for n different restriction endonucleases, the hybridization of n different target nucleic acids of different sequence with n different probe nucleic acids can be analyzed simultaneously or preferably in series.
• the method according to the invention can be carried out as follows: A DNA array of one or more probe nucleic acids according to the invention bound to a solid matrix is brought into contact with unlabelled sample nucleic acid, which can be RNA or DNA, under the preferred conditions listed below. The DNA array is incubated according to the surface to be hybridized with 20 ⁇ l - 200 ⁇ l of a suitable hybridization buffer at 45 ° C. for 10-20 minutes.
• 0.1 ⁇ g - 50 ⁇ g of unlabelled sample nucleic acid are taken up in 300 ⁇ l of a suitable hybridization solution and heated to about 99 ° C. for 5 minutes before hybridization with the DNA array and then cooled to about 45 ° C. for 5 minutes.
• the hybridization buffer is removed from the DNA array and replaced by the hybridization solution containing the sample nucleic acids.
• the DNA array is incubated for 16 hours at 45 ° C - 60 ° C with the sample nucleic acids. After removal of the sample nucleic acid solution, the DNA array is washed with washing buffers of different ionic strength, each at 50 ° C - 65 ° C.
• the activity of the restriction endonucleases can be increased up to 34-fold by adding lipids to the reaction mixture [Kinnunen et al., "Materials and methods for digestion of DNA or RNA using restriction endonucleases", US Pat. 5,879,950].
• the DNA array is corresponding to the surface to be hybridized with 20 ⁇ l - 200 ⁇ l of the reaction buffer recommended by the manufacturer, which contains 0.5 U - 5 U of the restriction endonuclease, which cleaves the probe nucleic acid in the region of the duplex ⁇ - ⁇ ', incubated for 20 - 60 minutes at 25 ° C - 37 ° C. Detached sample nucleic acids are obtained by washing with a 1X TE buffer at room temperature. Labels and restriction endonucleases removed from the surface of the DNA array The signals and data obtained by the method according to the invention are preferably detected and analyzed as follows:
• the concentration of the fluorophores conjugated with the probe nucleic acid is determined by measuring the absorption of an aqueous solution of the labeled probe nucleic acids at a wavelength which corresponds to the absorption maximum of the fiuorophores ( ⁇ la ) using the Lambert-Beer law. In order to be able to carry out a precise determination of the concentration of a nucleic acid conjugated with fluorophores, it must be taken into account that most fluorophores absorb light with a wavelength of 260 nm.
• the fraction of fluorophores is subtracted from the total absorption at 260 nm, which is a product of the absorption of the fluorophores at the wavelength corresponding to their absorption maximum ( ⁇ max ) and a correction factor CF 260 A 260 - (A ⁇ max x CF 260 )).
• ⁇ max absorption maximum
• CF 260 A 260 - A ⁇ max x CF 260
• the molar extinction coefficients of various fluorophores and the correction factors for the absorption at 260 nm (CF 260 ) are available from the manufacturers of these fluorophores (Molecular Probes, BioRad and others).
• the ratio of the concentration of the fluorophores conjugated to the probe nucleic acid to the concentration of the probe nucleic acid is equal to the amount of the fluorophores conjugated to the probe nucleic acid.
• the fluorescence of a defined amount of these probe nucleic acids is determined. Based on the specific fluorescence the number of probe nucleic acid molecules bound to a solid matrix can be precisely determined before hybridization with target or sample nucleic acid. Fluctuations in the amount of immobilized probe nucleic acids, which influence the hybridization with target or sample nucleic acids, can be taken into account in this way and corrected by calculation. Measurements that are carried out with the aid of the hybridization of target or sample nucleic acids with probe nucleic acids according to the invention can therefore be standardized.
• the fluorescence emission from each sample point of the DNA array is preferably determined by confocal laser scanning microscopy.
• Devices for determining the fluorescence emission on small areas are offered by a number of manufacturers and are laboratory standards in the field of biotechnology [Cheung, VG et al .; "Making and reading microarrays", Nature Genetics, vol. January 21, 1999; Bowtell, DDL, "Options available - from start to finish - for obtaining expression data by microarray", Nature Genetics, vol. Jan 21, 1999].
• the fluorescence of the immobilized probe nucleic acids is determined before hybridization.
• the fluorescence is excited by light of a wavelength that corresponds to the absorption maximum ( ⁇ A s . M a x ) of these fiuorophores, and detected at a wavelength that corresponds to the emission maximum ( ⁇ Em . ⁇ n a x ).
• n different probe nucleic acids of different sequence specificity which are labeled with n different, spectrally distinguishable fluorophores
• the fluorescence of the n different fluorophores is excited by light of a wavelength which corresponds to the absorption maximum ( ⁇ A b S . m a x ) corresponds to these fluorophores, and is detected at a wavelength which corresponds to the emission maximum ( ⁇ Em . m a ⁇ ).
• the determination of the fluorescence of different fluorophores can be carried out simultaneously or in succession.
• Twice modified oligodeoxynucleotides whose 5 'and 3' ends respectively of a C22 spacer with fluorescein isothiocyanate (FITC) or an amino group (NH 2) covalently linked (Sequence A: FITC-5 gcccgcgcAATAGGGATGGCTCAACAgcgcgggc 3 - (C22) NH 2 and B: FITC- 5 gcccgcgcTTAGAGTGCAAAATGAAAGCGCCgcgcgggc 3 - (C22) NH 2 ) were taken up in 100 ⁇ l coupling buffer (500 mM Na 2 HP0 4 , pH 8.5, 1 mM EDTA) (concentration: 500 pmol / ml).
• coupling buffer 500 mM Na 2 HP0 4 , pH 8.5, 1 mM EDTA
• Table 3 Fluorescence intensity x 1000 of the oligonucleotides covalently bound in the wells of a microtiter plate
• the wells of the microtiter plate were sheared with 125 ⁇ l hybridization solution containing 0.1 mg / ml Salmon Sperm DNA (Gibco BRL / Life Technologies); 0.5 mg / ml acetylated BSA (Gibco BRL / Life Technologies); 1X MES (100mM MES, 1.0M NaCl, 20mM EDTA, 0.01% Tween 20), prehybridized at 60 ° C for 4 hours.
• 1X MES 100mM MES, 1.0M NaCl, 20mM EDTA, 0.01% Tween 20
• Tables 2 Areas of the microtiter plate designated A ', B' and - were hybridized with the sample nucleic acids A ', B' or prehybridization solution The approach is for 20 min. heated to 95 ° C, cooled to 60 ° C over an hour and hybridized at 60 ° C for 16 hours.
• the wells of the microtiter plate were washed with "non-stringent” washing buffer (6X SSPE; 0.01% Tween 20) ten times for 5 minutes at 25 ° C. and then five times with "stringent” washing buffer (100 mM MES; 0.1 M NaCl; 0.01% Tween 20) for 5 minutes at 55 ° C. After washing, the wells of the microtiter plate were equilibrated with 150 ⁇ l 1X reaction buffer (NEB Buffer 3) for 10 minutes at 37 ° C.
• NEB Buffer 3 150 ⁇ l 1X reaction buffer
• 1X reaction buffer which contained 2 units of the restriction endonuclease Ach (New England Biolabs), for 1 Incubated at 37 ° C for one hour.
• the reaction was stopped by adding 1/5 volume stop solution (0.5% w / v SDS, 50 mM EDTA) and by heating the microtiter plate to 75 ° C.
• sample nucleic acids of the sequence A FITC- 5 gcccgcgcAATAGGGATGGCTCAACAgcgcgggc 3.
• B FITC- 5 gcccgcTTAGATGGcCc-GGcGcC-GGcGcCGGGcCc-GGcGcCGGGCCcGGcGcCGGGCCcGGCGcGcGcGcGcGcGcGcGcGccccc
• 5 ' gcccgcgcGTTTTTTTTTTTTTTGGTTTTTTTTTTTTC-gcgcgggc 3 '(control; determination of the background fluorescence) are sheared with 5 ⁇ g sample RNA, which was isolated from K562 cells, in 25 pM control RNA, 0.1 mg / ml Salmon Sperm B DNA / Life Technologies); 0.5 mg / ml acetylated BSA (Gibco BRL / Life Technologies); 1X MES (100mM MES, 1.0M NaCl, 20mM EDTA, 0.01 ' % Tween 20) hybridized at 55 ° C for 16 hours.
• 1X MES 100mM MES, 1.0M NaCl, 20mM EDTA, 0.01 ' % Tween 20
• the DNA array is washed ten times for 5 minutes at 25 ° C. with "non-stringent” washing buffer (6X SSPE; 0.01% Tween 20) and then five times with “stringent” washing buffer (100 mM MES; 0.1 M NaCl; 0.01% Tween 20) for 5 minutes at 50 ° C.
• non-stringent washing buffer 6X SSPE; 0.01% Tween 20
• stringent washing buffer 100 mM MES; 0.1 M NaCl; 0.01% Tween 20
• the DNA array is equilibrated with 1X reaction buffer for 10 minutes at 37 ° C.
• the DNA array is then incubated in 100 ⁇ l of 1X reaction buffer, which contains 2 units of the restriction endonuclease Aci1, at 37 ° C. for 1 hour.
• the reaction is stopped by adding 1/5 volume of stop solution (0.5% w / v SDS, 50 mM EDTA) and by heating the DNA array to 75 D C.
• the fluorescence of the hybridized probes remaining on the DNA array is determined by light of a wavelength ⁇ AS .
• the probe nucleic acids D (FITC) - 5 gcccgcgcG IIIIII TTTTTTGGTTTTTTTTTTTTTC-gcgcgggc 3 '
• E (Cascade Blue) - 5 gcc cgcgcGTTTTTTTTTTTTTTTTTTTC-gcgcgggc third
• F (BODIPY TR 14) - 5 gcccgcgc- GTTTTTTTTTTTTTTTTTTTTTC-gcgcgggc 3 ', which are used as a control for determining the background fluorescence, are immobilized together on a further defined area B (sample point B).
• cRNA sample RNA
• the DNA array is equilibrated with 1X reaction buffer for 10 minutes at 37 ° C.
• the DNA array is then incubated in 100 ⁇ l of 1X reaction buffer, which contains 2 units of the restriction endonuclease Aci1, at 37 ° C. for 1 hour.
• the reaction is stopped by adding 1/5 volume of stop solution (0.5% w / v SDS, 50 mM EDTA) and by heating the DNA array to 75 ° C.
• the signals detectable in the area of the sample point B correspond to the background fluorescence of the system and are subtracted from the signals detected in the area of the sample point A.

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  • 2002-11-25 WO PCT/EP2002/013215 patent/WO2003046215A1/de not_active Ceased
  • 2002-11-25 DE DE50207820T patent/DE50207820D1/de not_active Expired - Fee Related
  • 2002-11-25 JP JP2003547646A patent/JP2005536983A/ja not_active Ceased

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