DE10316159A1 - Analysis of nucleic acid by incorporation of luminescent dye during extension, useful especially for analysis of point mutations, is done on an optical waveguide chip in a flow cell - Google Patents

Abstract

Method for analyzing nucleic acid (NA) by incorporation of a luminescent dye (A) in presence of a polymerase, then detecting (A) on an optical waveguide chip, where incorporation of (A) is done in a flow cell. Method for analyzing nucleic acid (NA) comprises: (a) incubating, in a flow cell, (a mixture of) NA being analyzed with an enzyme (E) that can polymerize NA in presence of relevant co-factors; (b) polymerization of NA in presence of a luminescent dye (A) which becomes incorporated into, or bound to, the NA strand during polymerization; and (c) detecting polymerization by applying NA molecules, extended by polymerization, to an optical waveguide chip (X) and measuring the luminescent signal from incorporated (A). Independent claims are also included for the following: (1) device for the new process; (2) method for regenerating primers bound to a solid phase; and (3) method for analysis of point mutations.

Description

Nowadays, many nucleic acid analyzes are carried out on so-called DNA microarrays. These allow up to several 100,000 hybridizations to be measured simultaneously (De Vijver et al, 2002, New England Journal of Medicine Volume 347, No 25, pages 1999-2009). However, these measurement methods require complex sample preparation and a very complex probe design, since all probes on these chips must bind to the analyte in a specific manner at a constant temperature, ie only bind nucleic acid analytes with a homology of> 95%. It is therefore not surprising that many of these DNA microarrays give incorrect results, since it is almost impossible to calculate such a large number of probe molecules even halfway. One method to make the dependency of the analyzes on the melting point (Tm) of the nucleic acid probes independent is the analysis of DNA microarrays in temperature-controlled TIRF measuring devices ( EP 0001248948 ) such as the ATR reader developed by the Fraunhofer Society (FHG-IPM). This device works with prism chips into which the excitation light for fluorescence detection is guided in the chip by means of total reflection (optical waveguide chip). The excitation of fluorescent dyes using such optical waveguides is called TIRF (Total Internal Reflection Fluorescence). However, the ATR reader analysis method proves to be relatively complex since the melting curves of the nucleic acids are analyzed in order to determine the so-called melting points (Tm) of the nucleic acid hybrids ( DE 10309526.8 ). To do this, the melting points of DNA probes must be calculated and then experimentally determined. Another disadvantage of this method is that relatively large amounts of analyte are required to generate signals. This analyte is normally duplicated from DNA samples by PCR, if the analyte RNA, appropriate amplification mechanisms are used (eg RT-PCR). With these amplifications, the analyte is usually labeled at the same time, into which, for example, Cy5-dUTP is incorporated. A quantification of the original initial concentration of the nucleic acid analyte of individual nucleic acids is no longer possible after such an amplification. A competing method is the analysis of nucleic acids using a 5'-3 'exonuclease assay. Instead of parallelization, the focus is rather on quantifying the concentration of specific nucleic acids. For the real time PCR, probes are used for the quantification which are labeled with a fluorophore at the 5 'end and with a quencher at the 3' end. The probe attaches to the analyte during the annealing. During the extension, the nucleotide carrying the fluorophore is cleaved off by the 5'-exonuclease activity of the Taq polymerase by the Taq polymerase. The amount of fluorophore released into the liquid becomes measured quantitatively at the same time (corresponding procedural steps of PCR and real-time PCR are adequately described in the prior art and can be found, for example, in the following patents: US 4,683,202 ; US 4,683,195 ; US 4.9665.188 ; US 5,210,015 ; US 5,487,972 ; US 5,538,848 ). The combination of enzyme-mediated methods of molecular biology and DNA microarrays leads to applications such as solid-phase PCR (Shoffner et al., 1996, Nucleic Acids Research 24 (2): 380-385), primer extension and the so-called NOC -PCR ( EP 1186669 ). Although all of these methods are suitable for analyzing nucleic acids, they do not allow the content of a specific nucleic acid in the analyte to be determined even approximately.

On The main problem with the existing processes is the high effort involved the determination of nucleic acid concentrations, since these analyzes have so far not been parallelized, is also the automation effort is very high because none of the existing Systems over standardized fluidic interfaces. A major disadvantage the lack of quantifiability is smaller in chip-based systems Amounts of nucleic acid analytes, especially single copy RNA molecules.

The solution of the listed Problems arise in the simultaneous enzymatic duplication and labeling of the analyte nucleic acid and the detection of the synthesized new nucleic acids on a solid phase.

• a) Dem TIRF-Chip, in dem das Anregungslicht durch totale interne Reflektion geführt wird,
• b) der Flusszelle, die für Enzymreaktionen mit einer kompatiblen Polymerbeschichtung versehen ist und die in kurzen Taktzyklen geheizt und gekühlt werden kann und
• c) einer Detektionseinheit, vorzugsweise einer CCD-Kamera.

The invention describes the surprising finding that an enzymatic extension of nucleic acid probes can be measured in real time in a temperature-controlled and biomolecule-compatible coated flow cell, which is located on a TIRF chip. The system consists of three basic modules:

• a) the TIRF chip, in which the excitation light is guided by total internal reflection,
• b) the flow cell, which is provided with a compatible polymer coating for enzyme reactions and which can be heated and cooled in short cycle cycles, and
• c) a detection unit, preferably a CCD camera.

It is particularly important that the flow cell does not inhibit enzymatic reactions, such as this, by means of a suitable coating, such as, for example, polyethylene, polypropylene or polyethylene glycol eg takes place in metallic flow cells. At least one nucleic acid longer, complementary nucleic acid analyte is bound to immobilized nucleic acid probes, these serve as templates for an enzymatic extension of the immobilized nucleic acid probes and thereby generate a luminescence signal. The signal can be amplified by removing the nucleic acid template and attaching it again to an immobilized nucleic acid probe. According to the invention, the signal can be generated, for example, by incorporating luminescence-labeled nucleotide triphosphates (for example Cy5-dUTP). Luminophores according to the invention are, for example, but not exclusively: Cy-3, Cy-5, fluorescein, rhodamine, ROX, HEX, FAM, TAMRA, Amca. Luminance does not only include the effects of phosphorescence and fluorescence. However, other methods of luminescence labeling are also suitable, such as the coloring of nucleic acid double strands with intercalating dyes such as SyBr Green. Enzymes which are suitable for this method are, for example, but not exclusively: thermostable DNA polymerases, DNA polymerases in general, RNA-dependent DNA polymerases (“reverse transcriptases”). If the nucleic acid analyte is inadequate, this can be done by enzymatic This reaction can be carried out in parallel, the amplification of the template and the extension of the nucleic acid probe according to the invention taking place simultaneously in the same compartment of a TIRF apparatus. Methods for such an amplification are sufficiently known to the person skilled in the art PCR can be expanded exponentially, for example, RNA samples can be amplified by RT-PCR or by repeated reverse transcription with, for example, thermostable enzymes, and the increase in fluorescence on the sensor chip can also be measured at the same time urevorlage, as for example is also described for the 5'-3 'exonuclease assay. A strand of the analyte sought is attached to the immobilized probe during the annealing phase of the PCR and can thus serve as a template for an enzymatic extension. During the subsequent extension, there is not only a multiplication of the analyte in the supernatant, but also an extension of the immobilized probe via which the detection according to the invention is carried out. If not only “normal” nucleotide triphosphates are used, but also luminescence-labeled nucleoside triphosphates are used, the luminescence-labeled nucleoside triphosphates are also used to extend the immobilized probe. As a result, luminescent dyes accumulate in the evanescent field of the waveguide chip (sensor chip). By repeating the amplification luminescent dyes are further enriched on the immobilized probe molecules, and the increase in the signal per amplification cycle is a measure of the concentration of the analyte.

The heating and cooling of the analyte on the chip can take place, for example, in a temperature-controlled flow cell, as in the EP 0001248948 described. Other methods such as inductive heating or resistance heating also represent designs according to the invention. It is particularly important that the system is capable of heating and cooling. Cooling can be carried out not only by pelletizing elements, but also by other methods well known to the person skilled in the art, such as, for example, heat exchangers and cooling elements.

In a preferred embodiment of the invention, the oligonucleotide probes are on a solid or semi-solid surface to which they are covalently bound by means of a spacer. Spacer molecules with a length of at least 7 nm are particularly preferred, the spacer preferably consisting of a negatively charged or uncharged polymer. Polymers according to the invention are, for example, but not exclusively: polyethylene glycol, polythymidine, polyuracil, polyinosine polysaccharides and polyethylene oxide. However, copolymers and block copolymers can also be used. Polymers with a length of at least 10 monomers and / or at least 7 nm chain length are particularly preferred. Such polymers can be produced, for example, by immersing epoxy-reactive surfaces in polyethylene glycol and heating the immersed carrier to 60 ° C. for 6 hours. Other processes are well known to the person skilled in the art, such as, for example, “grafting-from” (WO 00/43539 A2) and “grafting-to” polymerizations ( EP 1132739 ).

Dyes according to the invention represent, for example, but not exclusively, luminescent dye conjugates Desoxyribonukleinsäuretriphosphate, Di-Desoxyribonukleinsäuretriphosphate, Ribonukleinsäuretriphosphate and their analogs, provided that these analogs are capable of Enzymes for chain extension of nucleic acid strands used to become. Can continue instead of luminescent dyes, so-called phosphorescent dyes be used. Such dyes can be complex, as molecules bound dyes or nanoparticles to the nucleotide triphosphates be bound. Can continue intercalating dyes such as SyBr Green, ethidium bromide used become.

Except for a simple flow cell with an inflow and outflow, as already in the DE 10002566 described, the flow cell according to the invention can be both compartmentalized and structures in the Show lumens of the flow cell. Compartments can, for example, be used to carry out molecular biological reactions immediately before analysis on the chip, such as polyerase reactions (PCR, reverse transcription) as well as other reactions such as exonuclease digestion (e.g. T7 Gen 6 exonuclease), but especially non-enzymatic reactions (e.g. chemical labeling). However, compartments can also have purely physical functions such as mixing liquids or tempering liquids. In particular, by applying a compartmentalized flow cell, the analysis area of the chip can be used to carry out several analyzes in parallel. In a particularly preferred embodiment, each flow cell can be controlled independently by its own inflow and outflow. Such a flow cell can consist, for example, of 8 compartments, two of which are arranged side by side and four are arranged one behind the other.

at Multiplex-PCR uses different primer pairs for amplification the analyte uses DNA. Different targets can be used here simultaneously to be analyzed. Multiplex PCR uses amplification of nucleic acids, as is well known to the person skilled in the art, correspondingly in PCR applications Manual, Boehringer published, carried out. there it may be that the individual primer concentrations of efficiency of the respective PCR system must be adjusted. The concentration the primer of very efficient systems relative to the primer concentrations less efficient systems degraded. If the PCR is on chip or carried out in a system upstream of the chip, so is the use of the uracil DNA glycosylase protocol (PCR Applications Manual, Boehringer) advantageous. The PCR instead dTTP dUTP built into the nucleic acid chain. Such molecules can using the enzyme uracil DNA glycosylase be dismantled. In this way, contamination in general be avoided. In a further embodiment according to the invention the PCR is carried out on chip or in an upstream compartment. To the system can decontaminate the compartments and the chip can be reused without having to replace parts.

at Hybridization to solid phases, especially when using single-stranded probes it is advantageous if the analyte is at least partially in the form of single-stranded nucleic acid molecules. According to the invention at least the single strand of the nucleic acid strand complementary to the probes. Usually, by melting the double-stranded DNA Single strands manufactured, here the renaturation reaction competes with hybridization on the solid phase probe. This can be done by a preferred amplification of the complementary Single strands can be bypassed by an asymmetric PCR. In a particularly preferred embodiment become the complementary single strands different target DNAs in an asymmetric multiplex PCR carried out. It goes without saying this method for common PCR methods, even if these are not explicitly listed here are suitable. Such methods, which are well known to the person skilled in the art are, for example, RT-PCR or DOP-PCR (PCR Applications Manual, Boehringer).

To an enzymatic extension of a solid phase primer it may be necessary to use the primer sequences to regenerate. For that there are different possibilities. Primer itself can be nuclease resistant, e.g. through the use of phosphorothioate primers or other modifications of the sugar residue. The through the polymerization added nucleotides can then removed by an exonuclease. Another method is the production of apyrimidinic sites through use by uracil instead of thymidine and removing the uracil from the DNA strand Uracil-DNA glycosylase. The apyrimidine sites are then identified by suitable enzymes (e.g. AP endonucleases) cleaved by 5'-OH residues for a new one Generate polymerization (Lindahl, T. 1982. "DNA Repair Enzymes" Ann. Rev. Biochem. 51: 61-87).

In a particularly preferred embodiment oligonucleotide probes are used in which pairs or n-tuples of probes that differ only in the last, 5'-base. Only in case of a match in this terminal area with the analyte there can be a polymerization come. This method is particularly useful for the detection of so-called "single nucleotides Polymorphisms "(SNPs) suitable. The investigation of such SNPs is e.g. B. for transplantation medicine (HLA analysis) and xenobiotic elimination (P450 isoenzyme analysis) are important.

example 1

The reverse transcription of the mRNA is carried out with the aid of a thermostable reverse transcriptase (Tth polymerase). 2–4μg RNA are melted in a flow cell of the ATR reader on a TIRF sensor chip in a 40μl sample batch (70 ° C, 10min). Standard reagents are added: 4μl 5-fold first strand buffer, 2μl 0.1M DTT, 1μl dNTP mix (10mM dATP, dCTP, dGTP, and dTTP dissolved in TE buffer, 10mM Cy5-dUTP) and 200 units of reverse transcriptase added. The specific primers are immobilized on the chip as oligonucleotide probes. The nucleic acids are with the reverse transcriptase on the immobilized probes elongated and the luminescent dye incorporated. To amplify the signal, the template DNA is melted again and the elongation step on the solid phase is repeated. The luminescence signal is measured and stored at each step.

Example 2

The Amplification of the analyte is carried out using specific primers and a Taq polymerase (Qiagen) performed. In 40μl Standard reagents are added to the sample mix: 1μl of the genomic sample DNA (500 ng / ul), 4μl standard PCR buffer, 1μl dNTP mix (10mM each of dATP, dCTP, dGTP, and dTTP dissolved in TE buffer, 10mM Cy5-dUTP), each 5μl specific Primer (200nm), 0.3μl Taq polymerase, the entire batch was treated with Aq. at least make up to 40μl. The complete sample mixture is in the flow cell at room temperature injected. Subsequently by heating to 94 ° C for 10min the inhibitor is cleaved from the Taq polymerase and the sample DNA denatured. The specific primers are as oligonucleotide probes immobilized on the chip. The nucleic acids are transferred to the with the Taq polymerase immobilized probes elongated and thereby the fluorescent dye built-in. The template DNA is melted again to amplify the signal and the step of elongation both on the solid phase as well in the supernatant repeated. The luminescence signal is measured at each step and saved. The heating-cooling steps are repeated 40 times.

Example 3

The Quantification of data using the 5'-3'-polymerase Assays obtained from Example 2 take place as follows. The fluorescence recordings from the measuring device are analyzed and the Fluorescence signals from the oligonucleotides are integrated by quantified. about the comparison with a data file becomes one of the measured points Assigned to a sequence. This data is after each Temperature step raised. In the case of signal generation takes the signal after each step. Unspecific signals indicate this Property not on. A graph is created from the respective signals creates the signal level against the number of cycles of amplification. From the data of these curves let yourself the original Determine the amount of DNA exactly. Appropriate methods are real-time PCR Technology well known to the expert and do not constitute mathematical Problems.

Example 4

to Processing large Sample quantities a pipetting robot is connected to the TIRF apparatus, which is able to take the samples from microtiter plates and to mix with the polymerase reagents. At the appropriate time the samples are taken from the microtiter plate at room temperature and with the pre-cooled reagents, shortly before the injection into the flow cell, mixed. The pre-cooled reagents are from a cooling compartment automatically fed to the apparatus. The mixing takes place in a mixing chamber by injection of the liquids and reciprocating along a Möbius mixer. After done Mixture, the reaction mixture is injected into the flow cell. The analysis is carried out as described in Example 2. Then will the apparatus with a surfactant-containing buffer, surfactant-free buffer and buffer rinsed with uracil DNA glycosylase. The equipment to 37 ° C heated. The resulting abasic sites on the DNA strand will be then cleaved by adding an AP endonuclease so that the oligonucleotide strands pass through again DNA polymerases extended can be. at Then demand becomes the TIRF chip is replaced. In the normal case, however, this is not necessary. The apparatus is now to the next Measurement ready.

Claims (18)

A method for analyzing nucleic acids comprising: (A) Incubation of a nucleic acid or a nucleic acid mixture to be analyzed with an enzyme capable of polymerizing nucleic acids in the presence needed Cofactors in a flow cell (b) polymerizing nucleic acids in Presence of at least one luminescent dye, which in the Polymerization in the nucleic acid strand is installed or attached. (c) Detection of the polymerization on an optical waveguide chip, with on the waveguide chip Nucleic acid molecules applied are, which are extended by the polymerization reaction, in which Nucleic acid molecules incorporated luminescent dyes or be attached and the luminescence signal of these dyes is measured. A method according to claim 1, characterized in that by repeating the polymerase step the signal generation elevated becomes. A method according to claim 2, characterized in that after each polymerase step the signal is measured and quantified becomes. Method according to one of the preceding claims, characterized characterized in that the polymerase is a thermostable DNA polymerase is. Method according to one of claims 1-3, characterized in that that the polymerase is RNA dependent Is DNA polymerase. Method according to one of the preceding claims, characterized characterized that the polymer reaction by heating-cooling steps carried out several times in succession becomes. Method according to one of the preceding claims, characterized characterized that the analysis is a quantification of the amount / concentration of the represents nucleic acid molecules to be examined, the increase in luminescence after each polymerization step is measured on the solid phase probe. Method according to one of the preceding claims, characterized characterized that the polymerase reaction in parallel through the use several primer sequences takes place in one compartment. Method according to one of the preceding claims, which the polymerase reaction preferably provides single-stranded nucleic acid molecules. Method according to one of the preceding claims, which the polymerase reaction is carried out in the presence of dUTP instead of dTTP and after the reaction, the reaction products using uracil DNA glycosylase be dismantled. Device for analyzing nucleic acids comprising: (A) Device for incubating a nucleic acid to be analyzed or of a nucleic acid mixture with an enzyme capable of polymerizing nucleic acids in the presence required cofactors in a flow cell (b) Device for polymerizing nucleic acids in the presence of at least one luminescent dye, which in the Polymerization in the nucleic acid strand is installed or attached. (c) Optical system for detection the polymerization on an optical waveguide chip, characterized in that that nucleic acid molecules are applied to the waveguide chip, which by the polymerization reaction can also be extended, being in this Nucleic acid molecules luminescent dyes be installed or attached and the luminescence signal this Dyes are measured by TIRF excitation. Device for analyzing nucleic acids after Claim 11 characterized in that it additionally has a system for evaluating and quantifying the measured luminescence signals Apparatus according to claim 11 or 12, wherein the Flow cell from at least two identical compartments for carrying out parallel Analysis exists. Apparatus according to claim 11 or 12, wherein the Flow cell with at least two different compartments different functions. Apparatus according to claim 11 to 14, wherein the Flow cell is coated with an inert polymer. Apparatus according to claim 11 to 15, wherein the TIRF waveguide with a polymer spacer of at least 7 nm in length is Process for the regeneration of solid phase bound Primer sequences include: (a) Extend the primers in the presence by dUTP (b) Treating the extended primer sequences with uracil DNA glycosylase (C) Cleavage of the abasic sites on the DNA strand for regeneration the primer sequence Method for analyzing point mutations thereby characterized that (a) On a waveguide chip, oligonucleotide probes which the bases to be examined complementary bases in their 5'-terminal positions have immobilized and (b) This to the sequence of nucleic acids to be examined are otherwise homologous and (c) These sequences on the TIRF Waveguide chips are hybridized with a nucleic acid analyte and (D) The probe sequence is then carried out on these hybrids using a DNA polymerase extended will and (e) The nucleic acid sequences formed using luminescent dyes is proven by TIRF