DE19518217A1 - Determn of DNA after capture by probe immobilised on solid phase - Google Patents

Abstract

Qualitative and quantitative determn. of DNA on solid phases comprises: (1) covalently bonding a single stranded DNA (capture probe) to a solid phase which has been chemically activated by treatment with an alcoholic soln. of organosilane then heating; (2) hybridising the complementary single-stranded DNA (which is to be detected) to the capture probe, and (3) the target DNA is determined: (a) directly from a marker already present in it; or (b) indirectly using an enzyme conjugate, anti-nucleic acid antibody or second DNA single strand (reporter probe) that hybridise with it. Also new is a device for this process comprising a shaped article having a surface, chemically activated by an adhesion promoter, covalently bound to a capture probe.

Description

The invention relates to a method and a Vorrich for the qualitative and quantitative determination of Deoxyribonucleic acid (DNA) on solid phases.

For the quantitative determination of DNA fragments Microtest plates as a solid phase became a variety of methods proposed (review by Guesdon, J. L., J. Immunol. Meth. 150, 1992, 33-49). These Methods have with the binding of the hybridized DNA to the test plates and the detection of the hybrids in the Cavities an analog final section. Of crucial Significance for the analytical sensitivity of this Methods is the binding of the nucleic acids to the Plate surface. As a result, the signal-to-noise Ratio significantly influenced.

The following techniques for binding nucleic acids Microtest plates have been described:

• - Binding of biotinylated DNA to avidin or Streptavidin-wetted plates;
• - binding of DNA to albumin-wetted plates;  
• - Binding of capture probes or target DNA to the Plate surface through passive adsorption.

So far, none of the methods mentioned has been able to Establish standard protocol. That is essentially because the adsorption-based binding ver do not expect high loading densities. In addition, the orientation of the molecules on the Plate surface is not defined and therefore a low specificity of the binding arises.

Other binding techniques based on covalent Bindings are described:

• - Formation of a phosphoamidate bond between the 5'- terminal phosphate group and Covalink® (Nunc) Microtest plates (Rasmussen et al., 1991: Anal. Biochem. 198, 138-142);
• - Covalent binding of aminated target DNA carboxylated micro test plates (Kohsaka et al., 1993: NAR 21, 3469-3472);
• - Binding of base-modified target DNA to a micro test plates (Keller et al., 1988: Anal. Biochem. 170, 441-450; Keller et al., 1989: Anal. Biochem. 177, 27-32);
• Covalent binding of target DNA to poly (Lys-HBr, Phe) -wetted plates, the wetting by Adsorption takes place (Running and Urdea, 1990: Biotechniques 8, 276-277).

So far, these methods have also failed don't enforce. They are for routine use too complex and too complicated. Are necessary for Example: incubation for several hours in ice or at 50 °. The micro test plates obtained are either not Can be stored or only in an airtight container Keep packaging.

In German patent specification DD 3 00 382 a Procedure for activating polystyrene solid body surfaces described. The activation of the Polystyrene surfaces are made with organosilanes. in the The result of this surface modification is obtained activated polystyrene surfaces with high binding ability and loyalty capacity. So it is according to this Patent specification possible to those activated in this way Polystyrene solid surface proteins such as antigens and antibodies, nucleic acids, low molecular weight Ligands, microorganisms, cells and other biological bind active materials. That happens in essential for separation and isolation corresponding Interaction partners and above all to the qualitative and quantitative detection of specific Interaction partners in the solid phase immunoassay. For Hybridization of single-stranded deoxyribo nucleic acids could be activated in this way Polystyrene surfaces are not used.

Detection of DNA fragments, in particular Polymerase chain reaction (PCR) amplificates are found extensive applications in medicine. So there is in the diagnosis or the follow-up of viral Infections and malignancies a strong one Need for quantitative statements, for example  Successful therapy or disease course in early To be able to judge stadiums. This diagnosis was so far not satisfactorily possible.

The invention is based on the object Offer detection methods for DNA and DNA fragments, the inexpensive, easy to use and in the existing laboratory equipment should be integrable as well as the possibility of automation should be left open.

The problem is solved with the characteristic Parts of claims 1 and 8.

It has surprisingly been found that the Transfer of enzyme immunological methods to Nucleic acid hybridization techniques meet the requirements on best meets. With nucleic acid Hybridization technology bind complementary Single strands of nucleic acids to form a Double strand (hybrid). The combination enzyme immunological methods with nucleic acid Hybridization techniques are carried out according to the invention Binding of the bases to a chemically activated solid Phase. At this stage there is single stranded DNA (Capture probe) now covalently bound and by in- Bringing a complementary to be demonstrated single-stranded DNA section (target DNA) Hybridization with the capture probe. Then will according to the invention, the determination of the target DNA by means of an egg target immunological determination method or via an antinucleic acid antibody, via insert a reporter sample or after prior marking the target DNA using a suitable assay (e.g. fluorometric or luminometric) performed. The  The solid phase is activated by treatment with organosilanes of the general formula

(XR ′ _{n ′} ) _n SiR _4-n

where X is an amino, carbonyl, glycidoxy, carboxy, Epoxy, halo, isocyano, diazo, isothiocyano, Nitroso, sulfhydryl or halocarbonyl group and R ′ are an alkyl, alkylphenyl or phenyl group, while R represents an alkoxy, phenoxy or halogen group stands, and n 'the value between 0 to 20 and n the value can have between 1 and 3.

The particularly advantageously used organosilanes be sit the general composition XCH₂CH₂CH₂- Si (OR) ₃ in the X for the reactive organic groups stands, which are listed above, and R for a methyl or Ethyl group.

Essential for the implementation of the invention Procedures are the conditions under which the The solid phase is activated. The activation takes place in alcoholic solutions with the following Warming, the activation being in aqueous Isopropanol, preferably in a ratio Isopropanol / water 1: 1, has proven. The following Heating is carried out according to the invention as far as it the physical properties of each allow solid phase (softening temperature). For Polycarbonate is heated up to 100-110 ° possible, for polystyrene, however, only heating up 55-65 °. This step makes it more surprising achieved that the single-stranded DNA (capture Probe) is bound, d. H. as many as possible DNA ends "point outwards" and thereby in Detection step a maximum hybridization possible is.  

In a further embodiment of the invention Claim 2 are molded bodies from natural as a solid phase Lichen or synthetic polymers used. As well it is possible to use molded articles made of glass. in the According to the invention, some are polysty ren, polycarbonate and polyvinylchloride and cellulose or cellulose-containing polymers. As a molded body especially the already known micro test plates or strips used. As a device for determination DNA on solid phases becomes a shaped body offered according to the invention, at its chemical activated surface single-stranded DNA covalent is bound such that hybridization with complementary single-stranded DNA sections possible is.

The covalent binding of the DNA to the invention fixed phases is an advantageous alternative to the above adsorptive and covalent methods. It shows that a specific bond with high Capacity and a strictly defined orientation of the Molecules occur on the solid phase surface.

The binding is made in a simple manner Protocol.

Further advantages of the invention are:

• - Little time required to bind the Capture-Son the;
• - Efficient binding of the capture probes, the one comparatively low use of probe DNA equip;  
• - Wide range of applications also with regard to the type of the solid phase and the detection method rens.

The invention is illustrated by the following examples and of the illustrations explained in more detail. Show it

Fig. 1 shows a time dependence of the hybridization of a synthetic target DNA in different capture probes (mean values from three experiments),

Fig. 2 an influence of the wetting concentration with capture probe 0-45 on the hybridization of a synthetic target DNA (mean values from four experiments),

Fig. 3 shows a linearity of hybridization in microtest plates from different manufacturers (mean values from experiments),

Fig. 4 shows an allele-specific hybridization of a synthetic sequence from exon 12 of the K-ras proto-oncogene depending on the washing temperature. The results of a typical experiment are shown, in which six different capture probes were used, which are complementary to the wild type (-GGT-) or to point mutations in the K-ras gene and

Fig. 5 shows a quantitative detection of amplification (typical experiment) - the product bands are shown in the agarose gel (bottom) and the corresponding luminometric signals for quantitative determination of the amplificates (top; see Table 3).

example 1 Activation of the plate surface

1. Silanization reagent NB 6130 (chemical plant Nünchritz, Nünchritz, Germany) was 1/5 in absolute ethanol diluted and in a volume of 50 µl pipetted onto the microtest plates per cavity. The Activator solution was added after an hour of incubation Room temperature removed, the plates for a further 2 hours dried at room temperature. In conclusion, the Cavities 3 × with 100 µl Aqua bidest. washed.

2. Microtitration plates made of polystyrene (Nunc, Denmark) with 20% glycidoxypropyltriethoxysilane (ABCR, FRG) in isopropanol / water (1: 1), pH 2.5, 1 h incubated at room temperature and then for 4 h Heated to 60 °. After washing three times with dist. The microtitration plates activated in this way become water incubated with the capture DNA.

3. Microtitration plates made of polycarbonate (Sarstedt, FRG) with a 20% mixture of aminopropyl triethoxysilane and glycidoxypropyltriethoxysilane (Hüls, FRG) in iso-propanol / water (1: 1), pH 2.5, at 1 h Contacted room temperature and after removal the excess solution heated to 110 ° for 4 h. To washing three times with dist. They will be water activated microtitration plates.

4. Polystyrene balls with a diameter of 6 mm are mixed with 10% glycidoxypropyltriethoxysilane (ABCR, FRG) in iso-propanol / water (1: 1), pH 2.5, at 1 h Incubated at room temperature and then at 60 ° for 2 h  warmed while constantly moving the balls. Inten washing with aqua dest. become the balls used.

Quantitative determination of a synthetic target DNA
The following experiments serve to describe basic test characteristics. The experiments were carried out with a 5′-biotinylated synthetic oligonucleotide (MWG Biotech, Munich, Germany) as the target DNA, the sequence from Yamada et al. (1991), Sample 2-121B, taken from:
5 ′ CAA-CGG-TGG-GGG-ATA-CAG-TTA-TGG-ATA-TTT-GGG-GAA-CGG-GAA-CTG-GTT-CGA-CCC-CTG-GGG-CCA-GGG-AAC 3 ′.

The capture probes were on an automatic DNA Synthesizer (Milligen, Burlington, USA) manufactured and via NAP 10 Sephadex G 25 columns (Pharmacia, Uppsala, Sweden) cleaned. The sequences of the capture probes are listed in Table 1.

Table 1

Capture probes for the detection of synthetic target DNA

Experiment 1 Time dependence of hybridization different capture probes

The length-different capture probes 0-26, 0-36, 0-45 and 0-57 were at a concentration of 40 µg / ml 2xPBS, pH 7.3 dissolved and in a volume of 50 ul Surface activated micro test plates (Polyha: MLW, Halberstadt, Germany). The probes were covalent over 2 hours of incubation at room temperature bound to the surface of the plate; subsequently the cavities 3 × with 70 µl PBS plus 0.2% Tween 20 pH 7.3 (hereinafter referred to as PBS / Tween) washed.

The target DNA was diluted in a concentration of 200 ng / ml in hybridization buffer (Tropix, Bedford, USA) and added to the plates in a volume of 50 μl. After different hybridization times between 1 and 60 minutes (see Fig. 1), the cavities were washed 3 times with 70 µl PBS / Tween.

Streptavidin-alkaline phosphatase conjugate (Tropix) was according to the manufacturer's instructions in conjugate pouf fer (Tropix) diluted in a volume of 50 µl given the hybridized oligonucleotides and 20 Mi incubated at room temperature. The samples were Washed 3 × with 70 µl PBS / Tween. Were used for detection 50 µl para-nitrophenyl phosphate in DEA buffer, pH 10 (Randox) pipetted onto the plates. The extinction at 405 nm became after 2 hours incubation at room temperature measured.  

The entire experiment was carried out in duplicate and was repeated twice.

Fig. 1 shows the results from which the dependence of the hybridization signal (readable from the extinctions) and the hybridization kinetics (readable from the time course) on the length of the capture probes becomes clear.

Experiment 2 Effect of wetting concentration with Capture probes

Capture probe 0-45 was used for covalent binding in 1 × PBS, pH 7.3 dissolved and in 6 different Concentrations from 0.2 ng to 20 µg / ml in one Volume of 50 Htl on activated micro test plates (Polysorp, Nunc, Roskilde; Denmark). To The cavities were 3 × for 1 hour incubation at 37 ° washed with 70 ul PBS / Tween.

The capture probes were labeled with 10 ng biotin Target DNA per cavity as indicated in Experiment 1 hybridizes.

For the luminometric detection of the hybrids, Incubate with the enzyme conjugate and three times Wa 50 µl assay buffer for alkaline phos phatase plus CSPD and 10% Emerald Enhancer (Tropix) pipetted the cavities. The samples were taken after 10 mi incubation at 37 ° in a luminescence reader (Luminoskan: Labsystems, Helsinki; Finland).

Fig. 2 shows the results of four experiments, each carried out in triplicate.

Experiment 3 Determination of linearity and the lower Detection limit

Micro test plates from different manufacturers (Polysorp, Maxisorp: Nunc; Microfluor: Dynatech, USA) were top surface activated and through covalent bonding with 100 ng / cavity capture probe 0-45 wetted. Hybridi sation and luminometric detection of the target DNA were done as described above. The target DNA was in geometric dilution in concentrations between 1 pg and 10 ng per cavity used.

Fig. 3 shows a summary of the results of six tests in quadruplicate. The calculated lower detection limit (blank plus 3 s) is 3.4 pg or 152 attomol.

Example 2 Allele-specific hybridization of a K-Ras sequence

The following experiments provide the basis for the detection of point mutations by allele-specific hybridization. As the target DNA, an oligonucleotide with the following sequence was synthesized from exon 12 of the K-Ras protooncogene:
5 ′ AGC-TGT-ATC-GTC-AAG-GCA-CTC-TTG-CCT-ACG-CCA-CCA-GCT- TCA-ACT-ACC-ACA-AGT-TTA-TAT-TCA-GTC 3 ′.

The oligonucleotide was purified on NAP 10 columns and according to the specifications of the manufacturer (Boehringer Mann home) using terminal deoxyribonucleotide Transferase labeled at the 3'-end with digoxigenin.

The capture probes were synthesized and purified as described. The sequences of the allele-specific Capture probes (Tab. 2) were Sasaki et al. (1990) taken. During capture probe 1 completely is complementary to the sequence of the target DNA the other probes each have a mismatch in the fourth triplet in front. The goal of the experiment was to create a Develop protocol with the full of incomplete complementarity can.

Table 2

Allele-specific capture probes for the detection of K-Ras Exon 12

Experiment 4

The allele-specific capture probes were in one Concentration of 20 µl dissolved in 2 × PBS, pH 7.3 and in a volume of 50 µl on surface activated Pipetted microtest plates (Polyha, MLW, Germany). The activation took place as indicated above. You probes were incubated at 37 ° for one hour, then the cavities were washed 3 × with 70 μl PBS each.  

50 μl of the conjugate buffer were used for prehybridization (Tropix) without enzyme addition for 20 min at 42 ° in cavities incubated; then the plates were 3 × each Wash 70 ul PBS / Tween. The target DNA was in a Concentration of 400 ng / ml in hybridization buffer (Tropix) recorded and for hybridization in a Vo lumens of 50 µl per cavity were incubated at 42 ° for 20 min.

The hybridized DNA was washed 3 × with 50 μl 0.1 × SSC (15 mM NaCl; 1.5 mM sodium citrate) plus 0.1% SDS stringently for 10 minutes. Temperatures between 42 ° and 50 ° were tested for this (see Fig. 4). The hybridized DNA was detected using an anti-digoxigenin / alkaline phosphatase conjugate (Boehringer, Mannheim). The conjugate was diluted 1/5000 in maleic acid buffer plus 10% blocking reagent (Boehringer, Mannheim). 50 ul enzyme conjugate per cavity were applied and for 30 min. incubated at room temperature. The cavities were then washed 3 × with 70 μl PBS / Tween each. For detection, 50 µl para-nitrophenyl phosphate in DEA buffer (Randox) were pipetted into each cavity. The absorbance measurement at 405 nm was carried out after 2 hours of incubation at room temperature. The experiment was carried out in triplicate.

Fig. 4 illustrates the relationship between the specificity of the measurement signal and the temperature for stringent washing.

Example 3 Quantitative detection of amplified Plasmid DNA

In the following experiment, the quantitative Be tuning of a target DNA after previous amplification tion shown by PCR. Use for amplification The plasmid DNA of the hybridoma line CB 03 became friends by Dr. S. Jahn, Institute for Medical Immunology, Charit´ Berlin provided.

Experiment 5

The plasmid DNA coding for antigen binding site 3 (CDR 3) was amplified according to Brisco et al. (1991). 200 nM digoxigenin-labeled VH-FR3A primers (5 ′ ACACGGC [C / T] - [G / C] TGTATTACTGT 3 ′), 200 nM biotin-labeled VH-JH primers (5 ′ TGAGGAGACGGTGACC 3 ′) (BioTez , Berlin, Germany), each 0.1 mM dNTP and 2.5 U / 50 .mu.l Taq polymerase (Boehringer, Mannheim, Germany) used. The plasmid DNA was used in a logarithmic dilution between 1 fg and 10 pg / 50 μl. The amplification was carried out after 9 min denaturation at 94 ° C over 30 cycles (1 min 94 °, 1 min 55 ° C, 1 min 72 ° C) in a model 9600 thermal cycler (Perkin Elmer). The amplificates were shown in a control electrophoresis in a 3% agarose gel ( Fig. 5).

17mer capture probes with the sequence (5 ′: GGT-ACT-TCT- GAC-CGG-GA 3 ′) (synthesis and settlement as above ) were in a concentration of 40 ul / ml dissolved in 1 × PBS, pH 7.3, in a volume of 50 µl / cavity on activated Polysorp micro test plates (Nunc, Roskilde, Denmark) pipetted and one hour  incubated at 37 ° C. Then the cavities Washed 3 × with 70 µl PBS / Tween each.

Streptavidin-wetted Dynabeads (Dynal, Oslo, Norway) were made according to the manufacturer's instructions with binding and washing buffer (BWP: 2 M NaCl, 10 mM Tris, 1mM EDTA; pH 7.5; Dynal) washed. 15 µl of PCR products were washed with 20 µl Magnetobeads with shaking for 30 min at room temperature incubated. After washing twice with BWP, the amplified products bound to the beads for strand separation taken up in 30 µl 0.1 M NaOH and at 20 min Incubated at room temperature. After one minute of fixation the beads in the MPCT (magnetic particle concentrator; Dynal) the supernatant was pipetted off with 15 µl 0.2 M HCl neutralized and in 45 µl 2 × Hybridization buffer (Tropix) diluted.

The single-stranded amplificates were in 2 aliquots each 40 µl divided on the wetted with capture probes Wells are pipetted and hybridized for 30 min Incubated at 42 ° C. The cavities were 3 × with 70 µl each PBS / Tween and 1 × with 70 µl maleic acid buffer plus Blocking reagent (Boehringer, Mannheim, Germany) washed. Then 50 µl of antidigoxigenin Alkaline phosphatase conjugate (Boehringer, Mannheim; 1/5000 in maleic acid buffer plus blocking reagent diluted) on the cavities and for 30 min Incubated at room temperature. After washing three times with 70 µl PBS / Tween were 50 µl assay buffer for Alkaline phosphatase plus CSPD luminogen and Emerald Luminescence amplifier (Tropix) on the samples pipetted and after 10 min incubation at 37 ° C Luminescence of the samples in the Luminoskan luminometer certainly.  

The values were calibrated by external experts Default.

Fig. 5 shows that the intensities of the product bands found in the agarose gel correspond to the amounts of amplification measured by luminometry. With reference to the external standard, the amplification efficiencies at the different initial concentrations of the samples could be determined (Table 3).

Table 3

Amplification efficiency depending on the starting amount of plasmid DNA

Claims (19)

1. A method for the qualitative and quantitative determination of deoxyribonucleic acid (DNA) on solid phases, characterized in that
that single-stranded DNA (capture probe) is covalently bound to a solid phase that is chemically activated by organosilanes in alcoholic solutions and subsequent heating,
that a complementary single-stranded DNA segment (target DNA) to be detected is hybridized with the capture probe
and that the target DNA is then determined either directly after prior labeling or indirectly via an enzyme conjugate, an antinucleic acid antibody or via a second single strand of DNA which hybridizes with the target DNA (reporter sample). 2. The method according to claim 1, characterized by that chemical activation in aqueous Isopropanol takes place.   3. The method according to claim 1 and 2, characterized by that the subsequent heating to below the Softening temperature of the one used solid phase. 4. The method according to claim 1 to 3, characterized by that as a solid phase molded from natural or synthetic polymers are used. 5. The method according to claim 1 to 4, characterized by that as polymers polystyrene, polyvinyl chloride, Po lycarbonate or cellulose-based polymers be set. 6. The method according to claim 1 to 3, characterized by that used as a solid phase molded body made of glass become. 7. The method according to claim 1 to 6, characterized by that polycarbonate is used and the after subsequent heating to 100-110 ° C takes place. 8. The method according to claim 1 to 6, characterized by that polystyrene is used and the warming at 55-65 °.   9. The method according to any one of claims 1 and 4 to 6, characterized by that as a shaped body micro test plates or strips be used. 10. The method according to any one of claims 1 and 4 to 6, characterized, spheres, tubes, polygons, etc. be used. 11. The method according to one or more of claims 1 to 10, characterized in that the solid phase is activated by means of organosilanes of the formula (XR '_n' ) _n SiR _4-n , where X is an amino, carbonyl, glycidoxy , Carboxy, epoxy, halo, isocyano, diazo, isothiocyano, nitroso, sulfhydryl or halocarbonyl group and R 'are an alkyl, alkylphenyl or phenyl group, while R is an alkoxy, phenoxy or halogen group, and n 'have the value between 0 to 20 and n have the value between 1 to 3. 12. The method according to any one of claims 1 to 10, characterized by that the activation of the solid phase with at least two organosilanes in a mixture or in succession liquid or gaseous phase. 13. A device for the qualitative and quantitative determination of deoxyribonucleic acids (DNA) on the most solid phases, characterized in that
that the device consists of a shaped body which has a surface chemically activated by means of an applied adhesion promoter,
that single-stranded DNA (capture probe) is covalently bound to this surface in such a way that hybridization with complementary single-stranded DNA sections (target DNA) is possible. 14. The apparatus according to claim 13, characterized in that the surface of the molded body as an adhesion promoter for the capture probes compounds of the general formula (XR '_n' ) _n SiR _4-n where X is an amino, carbonyl, glycidoxy, Carb oxy, epoxy, halo, isocyano, diazo, isothio cyano, nitroso, sulfhydryl or halocarbonyl group and R 'are an alkyl, alkylphenyl or phenyl group, while R is an alkoxy, phenoxy - Or halogen group, and n 'has the value between 0 to 20 and n have the value between 1 to 3, such that the binding to the surface of the molded body and to the capture probes takes place via the functional groups. 15. The device according to claims 13 or 14, characterized by that the molded body from natural or synthetic polymers exist.   16. The device according to one of claims 13 to 15, characterized, that as synthetic polymers polyvinyl chloride, Po lystyrene or polycarbonate can be used. 17. The device according to one of claims 13 to 16, characterized by that as natural polymers cellulose or cellulose-containing polymers can be used. 18. Device according to one of claims 13 or 14, characterized, that the moldings are made of glass. 19. Device according to one of claims 13 to 18, characterized, that the molded coatings made of polymers and Have glass.