DE10240941A1 - System for detecting nucleic acid, useful for analyzing mutations and single-nucleotide polymorphisms, comprises a support, with probes attached, that is used many times, sequentially - Google Patents

Abstract

Analytical system for detecting nucleic acid (NA) using a carrier, loaded with probe (capture) molecules, especially a biochip, for binding NA by hybridization, where the same carrier is used many times, sequentially, in molecular biology and/or chemical analyses. All or some of the analyses are the same or different and the carrier is subjected to standard methods for NA detection.

Description

The invention relates to an analytical, in particular diagnostic test system for sample nucleic acids under Use of a carrier equipped with nucleotide sequences, in particular biochips, which with the sample nucleic acids is loaded and on which the sample nucleic acids by hybridization the nucleotide sequences are fixed.

Carriers equipped with nucleotide sequences, in particular Biochip or bio microarray technology and in particular offer the DNA chip / DNA microarray technology a variety of uses in analytical tests (detection tests) and in perspective in diagnostic tests. Preferred areas of application include: Gene expression studies, sequencing, detection of microorganisms, and the detection of mutations or SNP (single nucleotide polymorphism). The implementation this - and other - tests / examinations / studies with the help of biochips or microarrays or the like with nucleotide sequences stocked carriers, in particular, DNA chips or DNA micro-arrays are also carried out regularly different methods, especially with hybridization methods (including the Hybridization with allele-specific oligonucleotides), DNA ligase reactions, Primer extension reactions, so-called mini-sequencing reactions, beacon hybridizations. There is always - if also in different degrees - the following disadvantages: (1) insufficient security with regard to specificity or reproducibility, (2) bad or restricted Suitability for high throughput tests - especially in If a large number of SNPs are detected simultaneously, (3) bad ones Suitability for the simultaneous investigation of several questions, for example the simultaneous analysis of expression, mutation and allele specificity, (4) partially high outlay on equipment and thus limited possible use.

The present invention lies based on the task of an analytical, especially diagnostic Test system for nucleic acids using or using carriers equipped with nucleotide sequences, in particular To create biochips with which these) disadvantages of the prior art are avoided are.

There is a solution to this problem in the provision of an analytical, especially a diagnostic Test system of the type mentioned, characterized in that is that one one and the same carrier (preferably chip) several times in succession in certain molecular biological and / or chemical analysis methods for nucleic acids, either all or some of these analytical methods are the same or different are, and that one concluding the chip a molecular biological and / or chemical detection method for nucleic acids.

• – es wird ein mit Nukleotidsequenzen als Sondenmoleleküle bzw. Fängermoleküle bestückter Träger, vorzugsweise ein Biochip/Microarray, insbesondere ein DNA-Chip/DNA-Microarray eingesetzt,
• – der Chip wird mit den Proben-Nukleinsäuren beladen,
• – die Proben-Nukleinsäuren werden im Zuge eines bekannten Hybridisierungsverfahrens an die Nukleotidsequenzen auf dem Träger bzw. Chip hybridisiert und damit auf diesem Träger/Chip fixiert,
• – der (ein und derselbe) Träger/Chip wird mehrmals nacheinander, d.h. zeitlich aufeinanderfolgend in bestimmten (definierten) molekularbiologischen und/oder chemischen Analyseverfahren für Nukleinsäuren einsetzt, wobei entweder alle oder einige dieser von dem Chip durchlaufenen Analyseverfahren gleichartig oder verschiedenartig sind.
• – der (selbe) Träger/Chip wird abschließend in einem molekularbiologischen und/oder chemischen Detektionsverfahren für Nukleinsäuren eingesetzt.

A molecular biological and / or chemical test system or analysis system for sample nucleic acids, in particular DNA and RNA sequences, is provided, which is characterized by the following features:

• A carrier, preferably a biochip / microarray, in particular a DNA chip / DNA microarray, is used, which is equipped with nucleotide sequences as probe molecules or capture molecules,
• The chip is loaded with the sample nucleic acids,
• The sample nucleic acids are hybridized to the nucleotide sequences on the support or chip in the course of a known hybridization process and thus fixed on this support / chip,
• - The (one and the same) carrier / chip is used several times in succession, that is to say successively in time in certain (defined) molecular biological and / or chemical analysis methods for nucleic acids, either all or some of these analysis methods passed through by the chip being identical or different.
• - The (same) carrier / chip is finally used in a molecular biological and / or chemical detection method for nucleic acids.

The term "(molecular biological and / or chemical) analysis method for nucleic acids " in the present context means performing one out of or multi-step chemical or biochemical, in particular molecular biological reaction, the product (reaction product) the information about the object or fact to be examined (analyzing) supplies. The term analytical test system or analysis system means here the combination of several defined molecular biological and / or chemical analysis methods such that the individual analysis methods (all or some of the same or different can) one after the other, i.e. consecutively on or with the one Object or object carried out on which the analytical question is based.

In the present context, the term “(molecular biological and / or chemical) detection method for nucleic acid” means a molecular biological and / or chemical method with which the correct sequence of the preceding analysis methods is confirmed. The person skilled in the art knows which method is suitable for this in each case. For example, a beacon hybridization, a ligase reaction, a primer extension reaction are suitable specific primer hybridization or various combinations of these methods.

The test system according to the invention ensures that this Result of an inserted (molecular biological and / or chemical) Analysis method checked by the following procedure and if necessary approved or affirmed becomes. This is where the respective advantages of the relevant processes come from to wear or to use and the possibly existing Disadvantages are compensated for. This way, overall it becomes a very high detection specificity achieved, several can Issues processed (examined) simultaneously on a carrier or chip and there is a possibility to carry out high throughput tests, e.g. the simultaneous investigation a big one Number of SNPs in an organism. The test system according to the invention is very special for the detection of certain known point mutations and SNPs suitable.

With the analysis methods mentioned can fundamentally at least around all previously known chemical and biochemical, especially molecular biological, act, and here especially those that are also traditionally already under Use of biochips performed were.

In a very simple embodiment of the test system according to the invention becomes the sample nucleic acids supporting beams, especially chip, first in a chemical ligase reaction and subsequently in a primer extension reaction. In this case, the primer extension reaction is also an analysis and detection methods.

Because the primer extension reaction Detection is only possible after ligation has taken place positive result of the primer extension reaction the confirmation that the previously performed Ligase reaction has ended.

A more complex embodiment of the test system according to the invention comprises preferably two or more analysis methods and a final detection method and is characterized in that the carrier or Chip with the on it, due to the expired analysis process extended Capture molecules and sample nucleic acids hybridized to it before the detection method in a denaturing process with subsequent washing process starts.

Through this intermediary Denaturation process with subsequent washing process the hybridized sample nucleic acids removed and the results (Reaction products) of the previously run analysis methods can be done in one or several subsequent detection methods can be confirmed. About that can out in the first analysis processes e.g. in the ligase reaction, specific labeled primer or specific in the primer extension reaction labeled nucleotides are used and these labels for detection can also be used.

For use in the test system according to the invention come all known carriers with nucleotide sequences as probe molecules (capture molecules) into consideration. Preferred carrier are all previously known and conceivable chip (basic) designs, in particular glass chips, nylon chips, nitrocellulose-coated chips, Plastic chips or so-called microelectronic chips.

The loading of the carrier or Chips with probe molecules (Capture molecules), i.e. the application and fixing of selected known and defined probe molecules (Capture molecules) takes place dependent on of the question and the analysis methods provided for the answer. As probe molecules (Catcher molecules) come in particular Oligonucleotides (modified or unmodified), PCR products, (specific) DNA molecules and RNA molecules (modified or unmodified).

A preferred variant of the analytical according to the invention Test system is suitable for the detection of point mutations and / or SNPs and provided and is characterized in that the nucleotide sequences the known sequence sections containing the mutation position of the nucleic acid samples complementary oligonucleotide probes are that the Mutation position is preferably at its 3 'end, and that it is either have the wild-type nucleotide or the mutate nucleotide.

The third to last base at the 3 'end of this Oligonucleotide probes are preferably a universal nucleotide analog trained to the specificity the ligase still increase.

This is another embodiment variant characterized in that at least one of the analytical methods using a ligase reaction of a specific primer.

A ligase is preferably used thermostable DNA ligase or a mutant form of a thermostable DNA ligase used.

A particularly suitable primer is characterized in that it has a nucleotide sequence which is composed (conceivable) of two sequence sections, namely the sequence section AI having the 3 'end and the sequence section A-II having the 5' end. The sequence section AI having the 3 'end is complementary to that section of the known sequence of the nucleic acid sample to be investigated which immediately follows the 3' end of the corresponding oligonucleotide probe sequence. The sequence section A-II having the 5 'end consists of one or more nucleotides, the nucleotide directly adjacent to the 5' end of the sequence section AI being identical to the last nucleotide at the 3 'end of the corresponding oligonucleotide probe, and the Sequenzab section A-II otherwise has no homology to the nucleic acid sample or oligonucleotide probe. In addition, the 5 'end of sequence section A-II can have modifications such as dephosphorylation or an additional linker.

When using such a primer is the ligase reaction with an additional enzymatic reaction coupled, in the course of which the sequence section A-II with the help specific enzymes such as thermostable flap endonucleases (FENs), including in particular Pfu and Afu FEN1 nucleases, or thermostable cleavases is cleaved from the primer.

Has proven to be a detection method in practice a ligase reaction using three different types Primers (K), (wt) (m) well proven. A Variant of these primers is characterized by the following nucleotide sequences: The nucleotide sequence of primer (K) is complementary to that Nucleotide sequence of the in the ligase reaction of the analysis method used primer, the nucleotide sequence of primer (wt) is complementary to that the nucleotide sequence containing the wild-type nucleotide at the 3 'end of the oligonucleotide probes, and the nucleotide sequence of primer (m) is complementary to that the nucleotide sequence containing the mutant nucleotide at the 3 'end of the oligonucleotide probes. Primers (wt) and / or primers (m) are preferably with a marker provided, by means of which the ligation product can be detected.

In particular, fluorescent dyes come as markers considering the additional possibility open, that primer (wt) and / or primer (m) with different fluorescent dyes are marked.

The marker can just as well be one additional Nucleotide sequence on one of the primers used, the after execution a ligase reaction, subsequent Heating (temperature increase), subsequent Washing process and final Rolling Circle Amplification (RCA) reaction is detected.

The invention is illustrated below of embodiments with associated Drawings closer explained. Show it

1 : an area (a spot, an area) of a chip / array that is loaded with an oligonucleotide probe,

2 : the area out 1 after adding a nucleic acid sample to be examined that hybridizes with the oligonucleotide probe,

3 : the area out 2 after adding a primer,

4 : the area out 3 after the ligase reaction has taken place, the primer is coupled to or ligated to the oligonucleotide probe,

5 : the area out 4 after adding a nucleotide mix (dATP, dGTP, dCTP, dTTP) and primer extension,

6 : the area out 5 after washing off the (all) nucleic acid samples to be examined from the oligonucleotide probe, subsequently carried out denaturation reaction and washing

7 : the area out 6 after adding a specific beacon.

8th : the area out 6 after adding different types of primers 8th' and 8th'' ,

9 : the area out 8th after hybridization and ligase reaction, the primers 8th' and 8th'' are on the oligonucleotide probes 4 with ligated primer 8th and DNA sequence / primer extension product synthesized thereon 10 bound.

Example 1: Combination of three analysis methods using a single DNA (bio) chip or DNA microarrays

To detect a particular mutation, e.g. G to A at position 495 of the XY gene, in a DNA sample (from a patient) becomes a ligase reaction with a primer extension reaction and a beacon hybridization coupled.

The procedure is as follows:

unterscheiden, und die jeweils komplementär zu dem bekannten, die Mutationsposition (z.B. Position 495 des Gens XY) enthaltenden Sequenzabschnitt der Nuleinsäureprobe 6 sind, d.h. die jeweils komplementär zu der Wildtyp-DNA-Sequenz bzw. der Mutations-DNA-Sequenz der Nukleinsäure-Proben 6 bzw. 6' sind. Infolge der Hybridisierung entstehen Hybride aus Oligonukleotidsonden 4, 4' und dazu komplementären Nukleinsäure-Proben 6, 6' aus der DNA-Probe des Patienten (vgl. 2). Im Idealfall werden dabei folgende Hybride gebildet: (a) Bei Homozygotie der Wildtyp-Sequenz entsteht an der Chipposition, die die zur Wildtyp-DNA-Sequenz komplementäre Oligonukleotidsonde 4 trägt, eine komplette Doppelstrang-DNA (2A). An der Chipposition, die die zur Mutations-Sequenz komplementäre Oligonukleotidsonde 4' trägt, entsteht eine Doppelstrang-DNA, die am 3'-Ende der Oligonukleotidsonde 4' einen Mismatch aufweist (2B). (b) Bei Homozygotie der Mutante entsteht die umgekehrte Situation, d.h. an der Chipposition, die die zur Mutations-Sequenz komplementäre Oligonukleotidsonde 4' trägt, entsteht eine komplette Doppelstrang-DNA (2A') und an der Chipposition, die die zur Wildtyp-DNA-Sequenz komplementäre Oligonukleotidsonde 4 trägt, entsteht eine Doppelstrang-DNA, die am 3'-Ende der Oligonukleotidsonde 4 einen Mismatch aufweist. (c) Bei Heterozygotie ist kein Mismatch vorhanden, d.h. an der Chipposition, die die zur Mutations-Sequenz komplementäre Oligonukleotidsonde 4' trägt, entsteht eine komplette Doppelstrang-DNA und ebenso an der Chipposition, die die zur Wildtyp-DNA-Sequenz komplementäre Oligonukleotidsonde 4 trägt.(I) First, a conventional DNA (bio) chip or DNA microarray 2 that with known oligonucleotides as capture molecules, for example with oligonucleotide probes 4 . 4 ' is equipped (see 1 ), used to carry out a nucleic acid analysis using a conventional hybridization reaction:
For this purpose, the patient DNA sample (to be analyzed) is placed on the DNA (bio) chip or DNA microarray 2 applied and the nucleic acid samples contained therein 6 then by hybridization to complementary oligonucleotide probes 4 bound. The following situation arises, for example, in a graphically simplified manner:
There are oligonucleotide probes in two positions on the chip 4 . 4 ' applied, which are only at their 3 'end in a base or a nucleotide

differ, and each complementary to the known sequence section containing the mutation position (eg position 495 of the gene XY) of the nucleic acid sample 6 are, that is in each case complementary to the wild-type DNA sequence or the mutation DNA sequence of the nucleic acid samples 6 respectively. 6 ' are. As a result of the hybridization, hybrids are formed from oligonucleotide probes 4 . 4 ' and complementary nucleic acid samples 6 . 6 ' from the patient's DNA sample (cf. 2 ). Ideally, the following hybrids are formed: (a) In the case of homozygosity of the wild-type sequence, the oligonucleotide probe which is complementary to the wild-type DNA sequence is formed at the chip position 4 carries a complete double-stranded DNA ( 2A ). At the chip position, which is complementary to the mutation sequence oligonucleotide probe 4 ' carries a double-stranded DNA, which is at the 3 'end of the oligonucleotide probe 4 ' has a mismatch ( 2 B ). (b) When the mutant is homozygous, the reverse situation arises, ie at the chip position, which is the oligonucleotide probe complementary to the mutation sequence 4 ' a complete double-stranded DNA ( 2A ' ) and at the chip position, which is the complementary oligonucleotide probe to the wild-type DNA sequence 4 carries a double-stranded DNA, which is at the 3 'end of the oligonucleotide probe 4 has a mismatch. (c) In the case of heterozygosity, there is no mismatch, ie at the chip position which is the oligonucleotide probe complementary to the mutation sequence 4 ' carries a complete double-stranded DNA and also at the chip position, which is the complementary oligonucleotide probe to the wild-type DNA sequence 4 wearing.

(II) Below is the same DNA (bio) chip or DNA microarray 2 with the hybrid (hybridization product) on it in a DNA ligase reaction with a specific primer 8th used, the sequence complementary to that section of the known sequence of the nucleic acid sample wild type 6 or the nucleic acid sample mutant 6 ' which is immediately the 3 'end of the oligonucleotide probe sequence 4 respectively 4 ' follows:
For this the chip 2 with the specific primer 8th offset (see 3 ) and the ligase reaction is carried out - either using thermostable ligases or chemically catalyzed - with the primer 8th to the oligonucleotide probe 4 is coupled or ligated to it (see 4 ).
Alternatively, the ligase reaction can be combined with an enzymatic reaction by using the thermostable endonuclease V.

If there is a mismatch at the transition from the 3 'end of the oligonucleotide probe 4 ' and the 5 'end of the primer ( 3B ) no ligation can take place.

This specificity regarding a mismatch is comparatively very high in the ligase reaction, e.g. far higher than in the primer extension reaction.

The reactions described under (I) and (II) represent the ideal case. In practice, however, there are a number of potential sources of error. Thus, although very rare, the ligase, for example, can perform a ligation despite the presence of a mismatch, particularly in the case of a TG or GT mismatch. Furthermore, for example, a homologous DNA sequence can be hybridized, particularly to the 3 'end of the oligonucleotide probe 4 is complementary. In this case an incorrect result would result. In addition, there is a risk that the primer sequence at its 5 'end will be complementary to another DNA sequence that is undesirably on the oligonucleotide probe 4 is hybridized, and a ligase reaction would also give a wrong result in this case.

With the test system according to the invention however, it is possible Identify such false results by both responses on the same sample nucleic acids performed the same chip by preferably at least one further analysis method (see step III) will, after that a denaturing and guard procedure (see step IV) and finally one or several detection methods (see step V).

(III) In a further (third) analysis method, the same DNA (bio) chip or DNA microarray is used 2 with the hybrid thereon and with it, namely to the oligonucleotide probe 4 ligated primer 8th used in a primer extension reaction:
For this the chip 2 mixed with a thermostable polymerase and a nucleotide mix (dATP, dGTP, dCTP, dTTP) and the primer extension reaction carried out (see 5 ). The reaction product is the oligonucleotide probe 4 with hybridized nucleic acid sample 6 on the one hand and ligated primer 8th and DNA sequence synthesized on it 10 (Primer extension product) on the other hand, where the synthesized DNA sequence 10 (the primer extension product) to a portion of the nucleic acid sample 6 which is not compatible with the oligonucleotide probe 4 hybridized, is complementary.

(IV) The same DNA (bio) chip or DNA microarray 2 with the hybrid on it to the oligonucleotide probe 4 ligated primer 8th and DNA sequence synthesized on it 10 (Primer Extension Product) is subjected to a common denaturation procedure and then one or more washing steps to obtain the nucleic acid sample 6 from the oligonucleotide probe 4 with primer attached to it 8th and DNA sequence synthesized on it 10 (Primer extension product) and detach from the chip 2 to remove (see 6 ). The chip 2 or the oligonucleotide probes fixed thereon 4 with primer attached to it 8th and DNA sequence synthesized on it 10 (Primer extension product) is / are then fed to a final detection method.

(V) In the detection process, the chip 2 (for example) with a beacon 12 which is only after specific hybridization with that to the oligonucleotide probe 4 ligated primer 8th and / or the DNA sequence synthesized thereon 10 (Primer extension product) emits a fluorescence signal so that a specific sequence recognition is indicated and at the same time the results of the previously performed analysis methods are confirmed.

This embodiment of the test system according to the invention has proven itself well in practice.

An advantageous modification of the test system described above is that in the primer extension reaction special thermo stable DNA polymerases and / or special additives which increase the specificity of the polymerase (s), for example thermostable Mut-S protein and / or the reagent "Perfect mismatch" (from STRATAGEN), and / or that Ligase reaction is carried out either enzymatically with thermostable ligase or chemically.

In another variant of the test system shown here by way of example, chips are used in which the probe oligonucleotides 4 . 4 ' are realized as RNA molecules. As a result, in the further course of the test system, RNA ligases are then used to carry out the ligation reaction, just as if RNA primers are used.

If the nucleic acid sample to be examined 6 is an RNA molecule, a (possibly thermostable) reverse transcriptase is used in the primer extension reaction instead of the DNA polymerase.

Are used as probe oligonucleotides Oligonucleotides with a "stem-loop" DNA structure can be used the ligase reaction takes place without the addition of primers.

For the detection of the respective reaction results the analysis methods used in the test system are all known and conceivable (standard) processes can be used individually or in combination, in particular the detection by means of fluorescent light, radioactivity, color reactions, Silver and / or gold particles.

There is also the possibility of modifying the test system described in such a way that individual reaction steps initially without or outside a carrier / chip / microarray 2 take place and the process is then continued on the chip / carrier.

For example, the ligase and primer extension reaction carried out in a reaction vessel and the hybrid afterwards applied to a chip and on a fixed oligonucleotide are hybridized so that its 3 'end can be ligated to the 5' end of the ligase primer. To The detection method then becomes a denaturing and washing step carried out.

Example 2: Investigation a patient's DNA sample for the presence of certain point mutations (A) according to the prior art and (B) according to the test system according to the invention.

A patient's DNA sample is said to diagnostic purposes for the presence of a particular point mutation to be examined, using the three known analytical methods: Hybridization with known oligonucleotides, primer extension reaction and ligase reaction.

(A) According to the prior art, the procedure is as follows:
Either (i) these analysis methods are carried out in the well-known manner using well-known carriers or reaction vessels, or (ii) biochips / microarrays are used instead of the well-known carriers or reaction vessels.

• 1. Die zu untersuchende DNA-Probe (des Patienten) wird beispielsweise mit Fluoreszenzfarbstoffen markiert und auf einen Chip aufgebracht, der mit spezifischen Oligonukleotiden bestückt ist, Anschließend wird eine im Stand der Technik geläufige Hybridisierungsreaktion durchgeführt und die dabei gebildeten Hybride mit dem Fachmann bekannten Verfahren detektiert.
• 2. Die zu untersuchende DNA-Probe (des Patienten) wird auf einen Chip aufgebracht, der mit spezifischen Oligonukleotiden bestückt ist. Nach Hybridisierung werden spezifische Primer zugegeben, die mit Fluoreszenzfarbstoffen oder radioaktiv markiert sind, und es wird eine im Stand der Technik geläufige Ligase-Reaktion durchgeführt. Das Reaktionsprodukt wird mit dem Fachmann bekannten Verfahren detektren.
• 3. Die zu untersuchende DNA-Probe (des Patienten) wird auf einen Chip aufgebracht, der mit spezifischen Oligonukleotidsonden bestückt ist. Anschließend wird eine im Stand der Technik geläufige Primer-Extension-Reaktion durchgeführt, bei der z.B. fluoeszenzmarkierte Nukleotide verwendet werden. Das markierte Reaktionsprodukt wird mit dem Fachmann bekannten Verfahren detektiert.

The procedure using chips essentially comprises the following steps:

• 1. The DNA sample to be examined (of the patient) is marked, for example, with fluorescent dyes and applied to a chip which is equipped with specific oligonucleotides. A hybridization reaction which is familiar in the prior art is then carried out and the hybrids formed in this way are known to those skilled in the art detected.
• 2. The DNA sample to be examined (of the patient) is applied to a chip which is equipped with specific oligonucleotides. After hybridization, specific primers are added which are labeled with fluorescent dyes or radioactively and a ligase reaction which is familiar in the prior art is carried out. The reaction product is detected using methods known to those skilled in the art.
• 3. The DNA sample to be examined (of the patient) is applied to a chip which is equipped with specific oligonucleotide probes. A primer extension reaction which is familiar in the prior art is then carried out, in which, for example, fluorescence-labeled nucleotides are used. The marked reaction product is detected using methods known to the person skilled in the art.

None of the individual analysis methods for themselves Taken, provides a result with a satisfactory level of diagnostic specificity Purposes.

To improve this specificity there are Possibilities, (a) each of the above steps with new chips two or more times perform and average the results obtained, or (b) the chip with one or more similar sets of specific ones Populate oligonucleotides to carry out several parallel approaches in one process run and to get a number of comparable results from which an average can be formed, or (c) the analysis methods mentioned are on one chip each, in this case three Chips, performed in parallel and the results with each other compared.

All of these options increase the meaningfulness of the ultimately determined result of the concerned Analysis procedures, but are costly, require the use larger quantities on patient DNA samples, and the specificity remains limited.

• 1. Ein Aliquot der zu untersuchenden DNA-Probe (des Patienten) wird auf einen Chip aufgebracht, der mit spezifischen Oligonukleotidsonden 4, 4' bestückt ist. Anschließend wird eine im Stand der Technik geläufige Hybridisierungsreaktion durchgeführt.
• 2. Auf denselben Chip werden spezifische Primer 8 aufgebracht und eine im Stand der Technik geläufige Ligase-Reaktion durchgeführt.
• 3. Derselbe Chip wird einer im Stand der Technik geläufigen Primer-Extension-Reaktion unterworfen.
• 4. Abschließend erfolgt ein Denaturierungs-Waschschritt und danach eine an sich bekannte Hybridisierungsreaktion mit einem spezifischen Beacon, dessen Nukleotidsequenz komplementär dem Übergang von dem anligierten spezifischen Primer und der durch die Primer-Extension-Reaktion verlängerten DNA-Sequenz ist. Das Fluoreszenzsignals des Beacon, welches nur nach spezifischer Hybridiserung des Beacons abgegeben wird, wird mit dem Fachmann bekannten Verfahren detektiert.

(B) According to the test system according to the invention, the procedure is as follows, for example:

• 1. An aliquot of the DNA sample to be examined (of the patient) is applied to a chip using specific oligonucleotide probes 4 . 4 ' is equipped. A hybridization reaction familiar to the prior art is then carried out.
• 2. Specific primers are applied to the same chip 8th applied and carried out a ligase reaction familiar in the prior art.
• 3. The same chip is subjected to a primer extension reaction known in the art.
• 4. Finally, there is a denaturing wash step and then a hybridization reaction known per se with a specific beacon, the nucleotide sequence of which is complementary to the transition from the ligated specific primer and the DNA sequence extended by the primer extension reaction. The fluorescence signal of the beacon, which is emitted only after specific hybridization of the beacon, is detected using methods known to those skilled in the art.

In contrast to the procedure according to the prior art in the procedure according to the invention (the test system according to the invention) the elected Analysis method for the first time in succession on the same chip carried out, whereby the control of process and results of the previous one Analysis procedure by the subsequent analysis procedure is given and the advantages of the respective analysis methods with regard to the specificity are combined. Through this internal control of the specificity of the previous past reactions) at any reaction point the chip gets the highest level of security and reliability in terms of of the test results guaranteed.

Example 3: Design variant of the test system according to the invention for examining a patient's DNA sample for presence certain and known point mutations and / or SNPs

(I) A conventional DNA (bio) chip or DNA microarray 2 with oligonucleotide probes / probe oligonucleotides 4 . 4 ' equipped, the third to last base at the 3 'end as a universal nucleotide analog (ie as a synthesized nucleotide which can base pair with each of the four nucleotides naturally occurring in the DNA dATP, dGTP, dCTP and dTTP) is formed).

An aliquot of the patient's DNA sample is applied to the chip and subjected to a hybridization process known per se. The nucleic acid samples contained in the patient's DNA sample and to be examined hybridize 6 . 6 ' to complementary oligonucleotide probes 4 . 4 ' ,

(II) Then the same DNA (bio) chip / DNA microarray 2 a DNA ligase reaction using a specific primer 8th subjected. The nucleotide sequence of the primer 8th is complementary to that section of the known sequence of the nucleic acid sample to be examined 6 . 6 ' which is immediately adjacent to the 3 'end of the corresponding oligonucleotide probe sequence 4 . 4 ' follows.

Due to the measure that the third last base at the 3 'end of the oligonucleotide probes 4 . 4 ' is designed as a universal nucleotide analog, the specificity of the ligase reaction is increased.

In order to further increase the specificity of the ligase reaction, it is proposed that in the publication by Jianying Luo et al., Nucleic Acid Res., 1996, Vol. 24, No. 14, pp. 3071-3078 to use the mutated Thermus thermophilus DNA ligase. We hereby expressly refer to the relevant content of this publication
Alternatively, the ligase reaction can be combined with an enzymatic reaction by using the thermostable endonuclease V.

(III) The same DNA (bio) chip / DNA microarray 2 from step (II), which is now the oligonucleotide probe 4 . 4 ' with ligated primer 8th and hybridized patient nucleic acid sample 6 . 6 ' is subsequently subjected to a primer extension reaction as described in Example 1 step (III).

(IV) The reaction product from step (III) is then subjected to a denaturing and washing process as described in Example 1 step (N), it still being according to the invention on the DNA (bio) chip / DNA microarray 2 located.

(V) The same DNA (bio) chip / DNA microarray 2 from step (IV), which now (only) oligonucleotide probe (s) 4 . 4 ' with ligated primer 8th and DNA sequence / primer extension product synthesized thereon 10 is finally subjected to a detection method, for example a beacon reaction as described in Example 1 step (V).

With this embodiment variant of the test system according to the invention are existing SNPs or point mutations with much greater specificity and reproducibility detectable as this using the methods known in the prior art possible is.

Example 4: Design variant of the test system according to the invention for examining a patient's DNA sample for presence certain and known point mutations and / or SNPs

(I) A conventional DNA (bio) chip or DNA microarray 2 is as described in step (I) of Example 1 or 3 with oligonucleotide probes / probe oligonucleotides 4 . 4 ' equipped and with the nucleic acid samples to be examined 6 . 6 ' (from the patient's DNA sample) using the hybridization method.

(II) Then the same DNA (bio) chip / DNA microarray 2 analogous to step (Π) of example 1 or 3 a DNA ligase reaction using a specific primer 8th subjected. The nucleotide sequence of the primer 8th is complementary to that section of the known sequence of the nucleic acid sample to be examined 6 . 6 ' that is immediately adjacent to the 3 'end of the oligonucleotide probe sequence 4 4 ' follows, but at its 5 'end there is another additional nucleotide which is identical to the last nucleotide at the 3' end of the oligonucleotide probe 4 ,

After hybridization of the primer 8th to the nucleic acid sample 6 . 6 ' consequently exists at the contact point between the 5 'end of the primer 8th and the 3 'end of the oligonucleotide probe 4 . 4 ' an "overhang" of a nucleotide. This "overhang" prevents the ligase enzyme from exerting its enzymatic activity. The ligase and the specific ligation between the oligonucleotide probe can only become effective after the use, for example, of a thermostable cleavase which enzymatically removes (cleaves) the "overhang" 4 . 4 ' and the primer 8th bring about (see. Anna V. Demchinskaya et al., J. Biochem. Biophys. Methods 2001, Vol. 50, pp. 78-89). The enzymatic cleavage usually only takes place if the additional nucleotide is identical to the last nucleotide at the 3 'end of the oligonucleotide probe 4 . 4 ' ,

The "overhang" can also comprise (be long) more than one nucleotide if necessary. This longer "overhang" (sequence section II) is realized, for example, by adding the above-mentioned additional nucleotide at the 5 'end of the primer 8th further nucleotides are synthesized, the resulting nucleotide sequence of each "overhang" (sequence section II) having no homology to the nucleic acid sample 6 . 6 ' or oligonucleotide probe 4 . 4 ' has (- in contrast to the sequence section I of the primer 8th ).

(III) The same DNA (bio) chip / DNA microarray 2 , which now carries the reaction product from step (II), is subsequently subjected to a primer extension reaction as described in example 1, step (III).

(IV) This is followed by this DNA (bio) chip / DNA microarray 2 and thus the reaction product from step (III) is subjected to a denaturing and washing process analogous to example 1, step (IV).

(V) Finally, this DNA (bio) chip / DNA microarray 2 together with the reaction product from step (IV) are subjected to a detection method, for example a beacon reaction according to Example 1 (V).

Example 5: Design variant of the test system according to the invention for examining a patient's DNA sample for presence certain and known point mutations and / or SNPs

(I) A conventional DNA (bio) chip or DNA microarray 2 is as described in step (I) of Examples 1 and / or 3 with oligonucleotide probes / probe oligonucleotides 4 . 4 ' equipped and with the nucleic acid samples to be examined 6 . 6 ' (from the patient's DNA sample) using the hybridization method.

(II) Then the same DNA (bio) chip / DNA microarray 2 analogous to step (II) of Examples 1, 3 or 4 of a DNA ligase reaction using a specific primer 8th subjected.

(III) The same DNA (bio) chip / DNA microarray 2 , which now carries the reaction product from step (II), can subsequently, as described in example 1 step (III), be subjected to a primer extension reaction.

(IV) This is followed by this DNA (bio) chip / DNA microarray 2 and thus the reaction product from step (III) is subjected to a denaturing and washing process analogous to example 1 step (IV).

(V) Finally, this DNA (bio) chip / DNA microanay 2 together with the reaction product from step (IV) (namely oligonucleotide probe 4 . 4 ' with ligated primer 8th and primer extension product synthesized thereon 10 but without a nucleic acid sample 6 . 6 ' ) subjected to a detection method. As such, here a ligase reaction analogous to Example 1 step (II) using three different types of primers, namely primers 8th' (K), primer 8th'' (wt) and primer 8th'' (m) performed.

primer 8th' For example, (K) is complementary to the primer used in step (II) 8th , ie its nucleotide sequence is complementary to the nucleotide sequence of the primer used in step (II) 8th and hybridizes with it. The two primers 8th'' (wt) and 8th'' (m) are complementary to the 3 'end of the oligonucleotide probes 4 . 4 ' , ie their nucleotide sequences are complementary to the nucleotide sequence at the 3 'end of the oligonucleotide probes 4 . 4 ' and consequently include the nucleotide position to be examined. primer 8th'' (wt) comprises the wild-type nucleotide and hybridizes with the corresponding oligonucleotide probe 4 , and primer 8th'' (m) comprises the mutant nucleotide and hybridizes with the corresponding oligonucleotide probe 4 ' ,

Alternatively, the nucleotide sequence is from primer 8th' (K) complementary to the sequence section adjacent to the wild-type nucleotide or mutant nucleotide at the 3 'end of the oligonucleotide probe sequence 4 . 4 ' , ie the last nucleotide at the 5 'end of the primer 8th' (K) is complementary to the penultimate nucleotide at the 3 'end of the oligonucleotide probes 4 . 4 ' , The two primers 8th'' (wt) and 8th'' (m) are complementary to the primer used in step II 8th . the last nucleotide at its 3 'end complementary to the last nucleotide at the 3' end of the oligonucleotide probes 4 . 4 ' is, that is complementary to the wild-type or mutant nucleotide of the oligonucleotide probes 4 . 4 ' is.

primer 8th'' (wt) and / or primer 8th'' (m) is / are provided with a marker by means of which the ligation product is detected.

Both primers are preferably fluorescence-labeled, with primers 8th'' (wt) with a different fluorescent dye as a primer 8th'' (m) is marked. The ligation product is then detected in a conventional manner using known and familiar methods. A heating, namely a treatment at elevated temperature, is preferably carried out beforehand, ie after the ligase reaction, the selected temperature being above the melting temperature of the (individual) primers used 8th . 8th' and 8th'' and below the melting temperature of the primers ligated together 8th - 8th' respectively. 8th - 8th'' , and then one or more washing steps are carried out. Because the respective positions of the wild-type nucleic acid sample 6 complementary oligonucleotide probes 4 and the mutant nucleic acid sample 6 ' complementary oligonucleotide probes 4 ' are known, the fluorescence color signals detected in the fluorescence microscopic detection must match the corresponding chip positions.

Another way of labeling the primers 8th'' (wt) and 8th'' (m) consists in equipping the primers with an additional nucleotide sequence which, after the ligase reaction, treatment with elevated temperature (heating) and washing process, enables a rolling circle amplification (RCA) reaction to be carried out with subsequent detection (cf. Girish Nallur et al., Nucleic Acids Res. 2001, Vol. 29, No. 23, e118).

Example 6: Design variant of the test system according to the invention for examining a patient's DNA sample for presence certain and known point mutations and / or SNPs

(I) A conventional DNA (bio) chip or DNA microarray 2 with oligonucleotide probes / probe oligonucleotides 4 analogous to step (I) of example 1 or 3 (namely only with one type of oligonucleotide probe / probe oligonucleotide) and with the nucleic acid samples to be examined 6 . 6 ' (from the patient's DNA sample) using the hybridization method. The position of the wild-type or mutant nucleotide in the nucleic acid sample 6 . 6 ' there is a few nucleotides from the 3 'end of the oligonuleotide probe in the present example 4 away.

(II) Then the same DNA (bio) chip / DNA microarray 2 with the hybrid (hybridization product) on it in a DNA ligase reaction with two specific primers 8th* (wt) and 8th* (m) used, their sequence complementary to that section of the known sequence of the nucleic acid sample to be examined wild type 6 or the nucleic acid sample mutant 6 ' which is immediately adjacent to the 3 'end of the corresponding oligonucleotide probe sequence 4 follows. primer 8th* (wt) is complementary to the relevant section of the nucleic acid sample wild type 6 and primer 8th* (m) is complementary to the relevant section of the nucleic acid sample mutant 6 ' , primer 8th* (wt) consequently contains in its sequence a few nucleotides from its 5 'end the complementary base to the wild-type nucleotide of the nucleic acid sample 6 and primer 8th* (m) contains in its sequence a few nucleotides from its 5 'end the complementary base to the mutant nucleotide. The primers are between this position and the 5 'end 8th* (wt) and 8th* (m) fluorescent labeled, the fluorescent label of the primer 8th* (wt) different from that of the primer 8th* (damn.

The chip 2 comes with the specific primers 8th* (wt) and 8th* (m) are added and the ligase reaction is carried out using a thermostable DNA ligase. In the course of this reaction, the primer 8th* (wt) or 8th* (m) to the oligonucleotide probe 4 coupled or ligated to this.

(III) Another analysis method can follow, in the course of which the same DNA (bio) chip / DNA microarray 2 with the hybrid thereon and with it, namely to the oligonucleotide probe 4 ligated primer 8th* (wt) or 8th* (m) is used in a primer extension reaction as described in Example 1 step (III).

(IV) Then, on the same DNA (bio) chip / DNA microarray 2 carried out an enzymatic reaction with the previous reaction product on it, in which a resolvase such as the enzyme T4 endonuclease VII or the thermostable endonuclease V is used. These enzymes specifically cleave mismatches.

(V) This is followed by this DNA (bio) chip / DNA microarray 2 and thus the previous reaction product is subjected to a denaturing and washing process analogous to Example 1, step (IV).

(VI) Finally, this DNA (bio) chip / DNA microarray 2 together with the previous reaction products (namely oligonucleotide probe 4 with ligated primer 8th* (wt) or 8th* (m), and if appropriate, ie when carrying out step (III) with the primer extension product synthesized thereon 10 and oligonucleotide probe 4 with the primer ligated and shortened by the enzymatic reaction in step (IV) and the denaturing and washing process in step (V) 8th* (m) and 8th* (wt), but without a primer extension product 10 and without a nucleic acid sample 6 . 6 ' ) subjected to a detection method. Beacon hybridization, a primer extension reaction or a ligase reaction are preferred for this Consideration. One of the primer sequences or Beaon sequences used is fluorescence-labeled and preferably complementary to the primer 8th* (wt) or 8th* (M). The fluorescent label is different from that of the primer 8th* (wt) and the primer 8th* (M). The resulting respective mixed colors can be assigned to the wild type or the mutant type: in the case of homozygosity for the wild type, only a certain mixed color can be detected and the monofluorescent color of the primers 8th* (M) tag. In the case of homozygosity for the mutant type, only a mixed color and the monofluorescent color are the primers 8th* (wt) marking detectable. In heterorygoty, both mixed colors and the two monofluorescent colors are the primers 8th* (wt) and 8th* (m) detectable.

All Variants described above of the test system according to the invention can in principle can also be combined with one another, in principle any one Combination comes into consideration. In particular, not only the complete Steps (I) to (V) of the different variants combined as desired become, namely for example step (I) from example 1, step (II) from example 3, step (III) and step (IV) from example 1 and step (V) from example 5, but it can also the corresponding steps of two or more examples can be combined, e.g. B. Step (II) from Example 1 with step (II) from Example 3, or step (II) from Example 4 with those described Primer modifications can be integrated in step (V) of Example 5 become.

Likewise, when using the carrier or Chips for specific transcript evidence of a ligase and primer extension reaction or using a primer extension reaction alone the respective detection reactions can be combined. To this Mutation and transcript detection can be carried out simultaneously, however, the possibility is given that initially ligase reaction only to detect the mutation and for then carry out the specific transcript verification for the primer extension reaction.

Substeps, especially the ligase reaction, can can also be repeated one or more times in succession, with a denaturation process or the ligase reaction before each repetition alternatively heating to a temperature that is above the melting temperature of the used (Single) primer is carried out should be.

2

DNA (bio) chip / DNA microarray

4

oligonucleotide probe

6

nucleic acid sample

8th

primer

10

Primer extension product

12

Beacon

Claims (20)

Analytical test system for sample nucleic acids using a carrier equipped with nucleotide sequences as probe molecules (capture molecules), in particular a biochip, which is loaded with the sample nucleic acids and on which the sample nucleic acids are fixed by hybridization to the nucleotide sequences, characterized in that one and the same carrier loaded with sample nucleic acids is used several times in succession in certain molecular biological and / or chemical analysis methods for nucleic acids, either all or some of these analysis methods being the same or different, and the carrier being finally used in a molecular biological and / or chemical detection method Subjects to nucleic acids. Analytical test system according to claim 1, characterized in that the Analytical methods of a chemical ligase reaction and a primer extension reaction are and that one the carrier first in the chemical ligase reaction and subsequently in the primer extension reaction starts. Analytical test system according to claim 1, characterized in that he the carrier before the detection process in a denaturing and washing process starts. Analytical test system according to one of claims 1 to 3, characterized in that that the detection method a beacon hybridization or a ligase reaction or a primer extension reaction or a specific primer hybridization or any combination of these methods. Analytical test system according to one of claims 1 to 4 for the detection of point mutations and / or SNPs, characterized in that the nucleotide sequences to the known sequence sections of the nucleic acid samples containing the mutation position 6 . 6 ' complementary oligonucleotide probes 4 . 4 ' are that the mutation position is preferably at its 3 'end and that they have either the wild-type nucleotide or the mutant nucleotide. Analytical test system according to claim 5, characterized in that the third to last base at the 3 'end of the oligonucleotide probes 4 . 4 ' is designed as a universal nucleotide analog. Analytical test system according to one of claims 1 to 6, characterized in that the or at least one of the analysis methods involves a ligase reaction using a specific primer 8th is. Analytical test system according to claim 7, characterized in that the Ligase a thermostable DNA ligase or a mutated form of a thermostable Is DNA ligase. Analytical test system according to claim 7 or 8, characterized in that that the Ligase reaction with an enzymatic reaction, especially under Use of the thermostable endonuclease V, is coupled. Analytical test system according to claim 7, characterized in that the primer 8th has a nucleotide sequence that is complementary to that section of the known sequence of the nucleic acid sample to be examined 6 . 6 ' which is immediately the 3 'end of the oligonucleotide probe sequence 4 . 4 ' follows, and which additionally has at its 5 'end a further nucleotide which is identical to the last nucleotide at the 3' end of the oligonucleotide probe 4 . 4 ' is. Analytical test system according to claim 7, characterized in that the primer 8th has a nucleotide sequence which consists of two sequence sections I and II, with sequence section I having the 3 'end of the nucleotide sequence and complementary to that section of the known sequence of the nucleic acid sample to be examined 6 . 6 ' which is immediately the 3 'end of the oligonucleotide probe sequence 4 . 4 ' follows, and wherein sequence section II has the 5 'end of the nucleotide sequence and consists of one or more nucleotides, the nucleotide adjacent to the 5' end of sequence section I being identical to the last nucleotide at the 3 'end of the oligonucleotide probe 4 . 4 ' is, and wherein the sequence section II otherwise no homology to the nucleic acid sample 6 . 6 ' and that the ligase reaction is coupled with an enzymatic reaction, in the course of which section II of the primer is cleaved using specific enzymes, preferably with thermostable flap endonucleases (FNEs), in particular Pfu and / or Afu FEN1 nucleases, or with thermostable cleavases becomes. Analytical test system according to one of Claims 7 to 11, characterized in that a ligase reaction using three different types of primers is used as the detection method 8th' (K), 8th'' (Wt) 8th'' (m) is performed using the nucleotide sequence of primer 8th' (K) complementary to the nucleotide sequence of the primer used in the ligase reaction of the analysis method 8th is the nucleotide sequence of primer 8th'' (wt) complementary to the nucleotide sequence containing the wild-type nucleotide at the 3 'end of the oligonucleotide probes 4 and the nucleotide sequence of primer 8th'' (m) complementary to the nucleotide sequence containing the mutant nucleotide at the 3 'end of the oligonucleotide probes 4 ' is, and being primer 8th'' (wt) and / or primer 8th'' (m) is / are provided with a marker by means of which the ligation product can be detected. Analytical test system according to one of claims 7 to 11, characterized in that a ligase reaction using various primers is used as the detection method 8th' (K), 8th'' (Wt) 8th'' (m) that the nucleotide sequence of primer 8th' (K) complementary to the sequence section adjacent to the wild-type or mutant nucleotide at the 3 'end of the oligonucleotide probes 4 . 4 ' is that the nucleotide sequence of primer 8th'' (wt) and primer 8th'' (m) complementary to the nucleotide sequence of the primer used in the ligase reaction of the analysis method 8th where the last nucleotide at its 3 'end is complementary to the last nucleotide at the 3' end of the oligonucleotide probes 4 . 4 ' is, and that primer 8th'' (wt) and / or primer 8th'' (m) is / are provided with a marker by means of which the ligation product can be detected. Analytical test system according to claim 12 or 13, characterized in that that the Marker is a fluorescent dye. Analytical test system according to claim 14, characterized in that primer 8th'' (wt) and / or primer 8th'' (m) is / are marked with different fluorescent dyes. Analytical test system according to one of claims 12-15, characterized in that after the Ligase reaction and heating is carried out before detection a temperature that is above the melting temperature of the used (Single) primer and below the melting temperature of the ligated together Primer lies, and that then a or several washing steps are carried out. Analytical test system according to claim 12 or 13, characterized in that the marker has an additional nucleotide sequence on one of the primers used 8th' (K), 8th'' (wt) or 8th'' (m), and that after the ligase reaction has taken place, heating, a washing process and a rolling circle amplification (RCA) reaction with subsequent detection are carried out. Analytical test system according to one of claims 1 to 17, characterized in that that one or several substeps, in particular the ligase reaction, are repeated one or more times, a denaturation step and / or before each repetition a warming to a temperature that is above the melting temperature of the used Primer is carried out becomes. Analytical test system according to one of claims 1 to 18, characterized that the carrier a Biochip or microarray is. Analytical test system according to claim 19, characterized in that the Chip a glass chip or nylon chip or one coated with nitrocellulose Chip or a microelectronic chip or a plastic chip.