EP1706505A2 - Procede pour valider et/ou calibrer un systeme pour l'execution d'experiences d'hybridation, jeu ordonne de micro-echantillons et necessaire correspondants - Google Patents

Procede pour valider et/ou calibrer un systeme pour l'execution d'experiences d'hybridation, jeu ordonne de micro-echantillons et necessaire correspondants

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Publication number
EP1706505A2
EP1706505A2 EP04804017A EP04804017A EP1706505A2 EP 1706505 A2 EP1706505 A2 EP 1706505A2 EP 04804017 A EP04804017 A EP 04804017A EP 04804017 A EP04804017 A EP 04804017A EP 1706505 A2 EP1706505 A2 EP 1706505A2
Authority
EP
European Patent Office
Prior art keywords
microarray
target molecules
hybrids
molecules
measuring point
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04804017A
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German (de)
English (en)
Inventor
Wolfgang Mann
Oliver Krispin
Petra HOFFMÜLLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alopex GmbH
Original Assignee
Alopex GmbH
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Filing date
Publication date
Application filed by Alopex GmbH filed Critical Alopex GmbH
Publication of EP1706505A2 publication Critical patent/EP1706505A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00693—Means for quality control

Definitions

  • the invention relates to a microarray, a kit and a method for validating and / or calibrating a system for carrying out hybridization experiments.
  • the invention relates to a DNA microarray, a corresponding kit and a corresponding method.
  • DNA microarrays The basic structure of DNA microarrays is evident, for example, from EP 476 014 B1, EP 619 321 B1, EP 386 229 B1 and EP 373 203 B1.
  • Capture probes are fixed to a substrate. Each capture probe has a specific sequence. From a sample mixture in which there are complementary strands of nucleic acids, individual strands of nucleic acids are extracted according to the key-lock principle by hybridizing with the complementary capture probes.
  • a system for carrying out hybridization experiments comprises a device, reagents (buffer, wash solution) and certain process steps (application of the sample, hybridization, washing).
  • DE 100 50 943 A1 describes a corresponding hybridization chamber in which a cyclic movement of the sample material on the array can be carried out by means of two adjacent vessels that communicate with the hybridization chamber.
  • EP 1 132 485 A2 describes a further hybridization device with a hybridization chamber.
  • This hybridization chamber has a transparent cover.
  • a device is provided with which the individual spots on the array are scanned through the transparent cover during the hybridization process.
  • a temperature control device is provided which is designed such that the array can be temperature-controlled at different temperatures. The temperature is varied during a measurement run and the array is scanned at different temperature values. This is intended to capture all spots under ideal conditions.
  • a further hybridization device emerges from EP 1 148 119 A1, in which the hybridization is to be influenced by means of an electric field.
  • hybridization chamber is part of a circuit in which a pump is arranged.
  • the sample material can be circulated continuously in this circuit so that the
  • Hybridization experiments are carried out with a constant flow of the sample material along the surface of the array.
  • the sample strands should be exactly complementary to the probes and have an exact setting of the physical conditions (temperature, salt, etc.). Correct in this context means that only exactly complementary strands are bound and that the reaction is in equilibrium. This is the only way to quantify analytically correctly, because the same number of Associate and dissociate partners. There is therefore a dynamic balance here. This means that the melting temperature Tm of the respective hybrid is set.
  • Such physical and chemical boundary conditions can on the one hand be device-specific features, such as the geometry of the hybridization chamber, or process-specific features, such as the temperature control on the array, possible agitation of the sample material, how the sample material is applied or how the array is rinsed, washed or dried.
  • the results may also depend on the buffer, salt used and the way in which the sample material and other reagents have been pipetted.
  • the signals can depend on further physical parameters, for example the flow rate (cf. US 2003/0162283) or the applied electrical field (cf. EP 1 148 119 A1). It is therefore practically impossible to compare experiments carried out with different devices or with different settings of the physical and chemical parameters, even if the same type of microarray should be used.
  • US Pat. No. 6,490,533 B2 discloses a scanner for sampling hybridization signals, the signals being normalized.
  • the normalization compensates for different amplitudes of excitation signals due to different optical properties of the scanning optics and the scanning electronics at different wavelengths.
  • WO 00/52625 describes a further normalization of data using software.
  • the invention is therefore based on the object of providing means which permit a validation or calibration of a system for carrying out hybridization experiments carried out by means of microarrays.
  • the object is achieved by a method with the feature of claim 1 or 2, by a microarray with the features of claim 3 and by a kit with the features of claim 17.
  • Advantageous embodiments of the invention are specified in the respective subclaims.
  • a microarray is used for validating and / or calibrating a system for carrying out microarray experiments, comprising: at least one first measuring point with probe molecules (PM) located therein, which are complementary to target molecules and which can form hybrids with the target molecules which have a melting temperature T m ( PM ),
  • At least one second measuring point with probe molecules (MM1) located therein which are incompletely complementary to the target molecules and which can form false hybrids with the target molecules, which have a melting temperature T m (MM i).
  • the method according to the invention further comprises the steps:
  • the parameter value at the maximum difference between the signal intensities of the first and second measuring points being the parameter value at which the system is approximately in equilibrium with regard to the hybrids in the first measuring point, and to which System is calibrated.
  • hybridization experiments are thus carried out under certain reaction conditions (device used, hybridization protocol used, etc.) in which target molecules are brought into connection with the probe molecules on the microarray which form hybrids or faulty hybrids. From the amount of hybrids and faulty hybrids formed in each case and their relationship to one another, it can be concluded whether the reaction conditions are suitable to meet the desired requirements for a hybridization experiment. If so, the reaction conditions are (or the system, device, method) validated. Comparing the results of different hybridization experiments that were carried out under different reaction conditions, one can select those reaction conditions in which the desired requirements are best met or at least meet a minimum standard.
  • the method according to the invention enables the calibration of a system under real test conditions.
  • the object is further achieved by a method for validating a system for performing microarray experiments with the following steps:
  • the validation being successful if the difference between the signal intensities of the first and second measuring points corresponds to a predetermined validation value.
  • the method according to the invention enables the validation of a system under real test conditions. It also enables different systems to be compared with one another.
  • microarray according to the invention for validating and / or calibrating a system for performing microarray experiments comprises:
  • PM PM measuring point with probe molecules located therein which are complementary to target molecules and which can form hybrids with the target molecules which have a melting temperature T m (PM).
  • MM1 An MM measuring point with probe molecules (MM1) located therein that are incompletely complementary to the target molecules and that with the target molecules
  • T m melting temperature
  • the microarray having a plurality of PM measuring points with different melting temperatures and corresponding MM measuring points and the different ones Cover melting temperatures of at least 15 ° C.
  • the microarray according to the invention also has: at least one second MM measuring point assigned to a specific PM measuring point with probe molecules (MM2) located therein which are incompletely complementary to the target molecules which can form false hybrids with the target molecules which form distinguish the probe molecules of the other MM measuring point corresponding to the PM measuring point and which have a melting temperature T m (MM2)
  • a further advantageous embodiment of a microarray according to the invention is characterized in that the probe molecules are formed from: DNA, RNA, mRNA, cDNA, PNA, tRNA, mRNA, LNA, aRNA, PNA, proteins, antigens / antibodies, peptides, steroid hormones or others biologically relevant analytes
  • a further advantageous embodiment of a microarray according to the invention is characterized in that the probe molecules are formed from oligonucleotides produced ex situ.
  • Such probe molecules can easily be provided in pure form, which is why the homogeneity of measuring points can be easily guaranteed. Their use therefore leads to high quality microarrays in an economical manner.
  • thermo difference .DELTA.T is between the melting temperature Tm (PM) of hybrids and the melting temperature T m (MM1) and / or Tm (MM2) of incorrect hybrids at least 0.5 ° C.
  • Such a microarray enables a particularly precise and convenient calibration or validation of a system for performing microarray experiments.
  • Further advantageous embodiments of a microarray according to the invention are characterized in that the temperature difference ⁇ T between the melting temperature Tm (PM) of hybrids and the melting temperature T m (MM1) and / or T m (MM2) of faulty hybrids between 0.1 ° C and 5 ° C.
  • a preferred embodiment of the microarray according to the invention is characterized in that each measurement point on the array occurs several times.
  • microarray enables local differences in hybridization reactions to be recognized as a function of the reaction conditions when calibrating or validating a system.
  • a configuration is also advantageous in which the PM measuring point is in the immediate vicinity of the MM measuring point (MM1) or the further MM measuring point (MM2).
  • a microarray of several PM measuring points preferably more than 2 or 3 PM measuring points, enables calibration methods or validation methods to be carried out at a comparatively large temperature range.
  • Such a microarray is advantageously further characterized in that the difference in the melting temperatures of hybrids of the target molecules with complementary probe points of different PM measuring points is at least 1 ° C. to 10 ° C.
  • Such a microarray is also advantageously characterized in that the different melting temperatures of the different PM measuring points unite Cover the range from at least 20 ° C to 60 ° C.
  • kits for validating and / or calibrating systems for carrying out microarray experiments comprising: a microarray according to the invention, and
  • the probe molecules (PM, MM) and the target molecules are formed from: DNA, RNA, mRNA, cDNA, PNA, tRNA, mRNA, LNA, aRNA, PNA, proteins, antigens / Antibodies, peptides, steroid hormones or other biologically relevant analytes
  • a further embodiment of the kit according to the invention is characterized in that the temperature difference ⁇ T between the melting temperature Tm (PM) of hybrids and the melting temperature Tm (MM) of faulty hybrids is at least 0.5 K.
  • FIG. 1 schematically shows three possible states of a hybridization equilibrium between probe molecules and target molecules, and signal intensities resulting therefrom,
  • FIG. 2A shows a schematic representation of the structure of probe molecules of a microarray according to the invention
  • FIG. 2B shows a schematic representation of the structure of further probe molecules of a microarray according to the invention
  • FIG. 3 shows a table listing the probe molecules of a microarray according to the invention
  • FIG. 4A shows a schematic representation of the structure of a target molecule according to the present invention
  • FIG. 4B shows a schematic representation of the structure of further target molecules according to the invention
  • FIG. 5 shows a table in which different target molecules according to the invention are listed
  • FIG. 6 shows a schematic representation of the signal intensities in the measuring points of a plurality of microarrays according to the invention after carrying out a method according to the invention
  • FIGS. 7A to 7D each have tables which relate to one of the microarrays shown in FIG. 6 at different temperatures
  • FIG. 8 shows a hybridization protocol
  • Figure 9 is a table listing the probe molecules of a microarray according to the invention and the corresponding different target molecules.
  • Microarrays have a substrate 1, on which probe molecules 2 are arranged, which can form a hybrid with target molecules 3.
  • the probe molecules 2 on the substrate 1 are grouped into measuring points, which are also referred to as spots.
  • PM first measuring point or PM measuring point with probe molecules
  • Tm melting temperature
  • MM1; mm mismatch
  • False hybrids are hybrids that are formed from a probe molecule and a target molecule that are not exactly complementary. If the probe molecules and the target molecules are formed from oligonucleotides or other sequences, the faulty hybrids typically have one or two defects. However, the target molecules and the probe molecules do not have to be a sequence. For example, they can also be formed from antigens and corresponding antibodies, which can also form a hybrid according to the key-lock principle. In this case, faulty hybrids are formed by probe molecules that do not exactly match the target molecules, but whose deviation is so small that hybridization is also possible when the melting temperature Tm (PM) is reduced.
  • Tm melting temperature
  • a faulty hybrid can therefore also be defined by its melting temperature T m (MM), which is at least 0.5 ° C lower than the melting temperature T m (PM) of the hybrid of complementary target molecule and probe molecule.
  • the melting temperature T m (MM) is preferably less than 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C. or 10 ° C. than the melting temperature T m (PM).
  • Tm the amount of hybrids corresponds to the amount of free probe molecules. This fact is indicated by reaction arrows of the same length. If the experiment is interrupted at this point, a signal intensity la is obtained in the probe point in which the probe molecules 2 are located.
  • the hybridization reaction proceeds faster than the dissociation reaction, and at least theoretically, this should result in more hybrids than single molecules, which should lead to a higher signal intensity purple in the corresponding measurement point. This does not always have to be the case, since target molecules can be withdrawn from the reaction mixture due to incorrect hybridizations with other probes.
  • this temperature T 3 is closer to the melting temperature T m (MM) of the faulty hybrids than T 2 or Ti, and then higher signal intensities are obtained for the faulty hybrids than in the other two experiments.
  • the signal intensity of the hybrid signal does not increase to the same extent as the signal intensity of the faulty hybrid signal, especially since the formation of faulty hybrids removes target molecules from the reaction mixture which are then no longer available for hybrid formation. As a result, the distance between these two signal intensities decreases.
  • the difference between the signal intensities of measuring points which have hybrids and of measuring points which have faulty hybrids is therefore greatest at a temperature Ti which corresponds to the melting temperature T m of the hybrids.
  • the respective hybridization experiment is carried out at a temperature which is approximately the melting temperature T m of the conceivable hybrids between the probe molecules present on the microarray and the matching target molecules. Only if physical and chemical parameters are observed that allow the establishment of a chemical equilibrium at which the dissociation rate is at least approximately the association rate, is the amount of detectable incorrect hybridizations small compared to the amount of detectable hybridizations, and only then can make reliable qualitative and, if necessary, quantitative statements or statements about the proportions of target molecules in a biological sample.
  • the calculated melting temperatures of different probe molecules (or their hybrids with target molecules) on a microarray are generally as identical as possible or are at least in a very narrow temperature range.
  • This temperature is generally calculated using prior art methods as outlined above. These calculations are based on idealized conditions and it is not possible to carry out these calculations so precisely that all influences that can affect the respective hybridization experiment are included in the calculation. This includes a large number of parameters, for example the pressure prevailing in a hybridization chamber, the geometry of the hybridization used. chamber, the manner in which a reaction mixture is applied, the composition of the reaction mixture (concentration of target molecules, buffer contained therein, pH value, etc.), the reaction time, etc.
  • the microarray according to the invention makes it possible to carry out a calibration process in which the microarray is associated with target molecules which are complementary to the probe molecules in the first measuring point of the microarray, and a specific parameter which is to be calibrated is used in the hybridization experiments is calibrated.
  • the parameter value at the maximum difference between the signal intensities of the first and second measuring points is, for the reasons mentioned above, the parameter value at which the system is approximately in equilibrium with regard to the hybrids in the first measuring point, that is to say at which the system reaches a state which corresponds approximately to that of the reaction equation I in FIG. 1.
  • the temperature at which a microarray experiment is carried out depends on the probes arranged on the respective microarray and the resultant optimal optimal hybridization temperature for such a microarray, it is of particular advantage if one can use a microarray according to the invention which has several sets of measuring points, each with at least a first measuring point with complementary probe molecules (PM) and a second measuring point with the Target molecules have incompletely complementary probe molecules (MM), the hybrids of the target molecules with the complementary probe points (PM) of the different sets each having a different melting temperature, and wherein for each set separate probe and target molecules are provided, which are selected in such a way that incorrect hybridizations or cross-hybridizations between target molecules of one set and probe molecules of another set are almost impossible.
  • MM incompletely complementary probe molecules
  • the difference in melting temperatures of hybrids of the target molecules with complementary probes of different sets is at least one degree Celsius, and with the aid of these sets a range between 20 ° C and 60 ° C is covered, the user can use such a microarray to systems for performing hybridization experiments that run at any operating temperature between 20 ° C and 50 ° C.
  • the manufacturer of a hybridization device in which the hybridization reaction is to run fully automatically can use parameters such as the type of agitation of the hybridization chamber, the applied layer thickness of the sample material, the reaction time, etc. for every conceivable operating temperature between 20 ° C and 60 ° C using the microarray optimize and store in a device memory that is connected to a controller that controls the device taking into account the data contained in the memory.
  • the end user then only has to enter the hybridization temperature desired by him, and the device can use the controller to automatically set the parameters which are most favorable for the hybridization temperature selected in each case.
  • hybridization devices can also be recalibrated, for example by the manufacturer, or else checked or recalibrated by the user himself.
  • a system is validated by connecting the microarray to target molecules at about the melting temperature of the hybrids and carrying out the hybridization according to a predetermined protocol (see FIG. 8) and then determining whether the difference between the signal intensities of the first measuring points with hybrids and of the second or third measuring points with faulty hybrids corresponds to or exceeds a predetermined value. This value is to be specified for each device type by the manufacturer of the device. If a system fulfills this condition, it is validated correctly. Otherwise the device does not meet the requirements.
  • Different systems that is to say devices and / or methods for hybridizing, can be compared with one another on the basis of the values determined.
  • the validation can be carried out separately with the aid of a microarray according to the invention with different sets of probe molecules at different for each melting temperature for which a set is provided on the microarray.
  • Calibration and validation can be repeated at different points on a microarray at the same time.
  • the first, second and possibly third measuring points of a set of probe molecules can be repeatedly applied to a microarray at different locations.
  • a microarray can thus be completely covered by measuring points of one or more sets that are repeated at regular intervals.
  • inhomogeneities can be easily detected by means of such a microarray: at the points where the temperature of the microarray T m comes closest, the detected signal intensities of the respective measuring points of a set have the highest values and at other points lower values.
  • those parameters can be determined in which the differences between the values determined in this way of otherwise identical sentences are reduced to a minimum.
  • the invention is explained in more detail below in the context of an exemplary embodiment.
  • oligonucleotides are used as probe molecules and target molecules. However, this is in no way to be understood as limiting.
  • probes and target molecules such as DNA, RNA, mRNA, cDNA, PNA, tRNA, mRNA, LNA, aRNA, PNA, proteins, antigens / antibodies, peptides, stereoid hormones or other biologically relevant analytes.
  • Measuring points are applied at regular intervals on an epoxy-coated glass slide (75x25 mm Schott nexterion slide E), in which oligonucleotides with a total length of 26 or 30 nucleotides and with an amino link at the 5 'end are provided.
  • the oligonucleotide sequences each contain one
  • the relevant sequence for the hybridization is a 16 or 20mer.
  • the hybridization-relevant sequence is composed of 4 or 5 blocks of four nucleotides each. Two of the four blocks determine the GC content of the oligonucleotide and thus essentially its melting temperature T m , the remaining blocks have a GC content of 50%.
  • FIGS. 2A, 2B A schematic illustration of such probes is shown in FIGS. 2A, 2B.
  • 2A shows the schematic structure of a probe called GC 67, the melting temperature of which is 67 ° C.
  • 2B shows the schematic structure of two further probes GC 58 and GC 48, each of which has a melting temperature of 58 ° C. or 48 ° C.
  • probes on the microarray which differ from the probes by one nucleotide (mismatch 1, MM1) or by two nucleotides (mismatch2, MM2).
  • FIGS. 2A, 2B the respective four-nucleotide block in which such a nucleotide was exchanged is shown schematically with the designations MM1, MM2.
  • the exact sequences of the probes provided on the chip as well as their designation can be seen from the table in FIG. 3.
  • the nucleotide exchange takes place in the four-nucleotide block, which defines the GC content of the respective microarray probe.
  • the GC content of the microarray probe molecule is not changed by the nucleotide exchange, the GC content of the mismatch probe molecules corresponds to the GC content of the associated perfectmatch Probe molecules.
  • the spotted chipset also contains a negative control (GC 37).
  • Target molecules are synthesized for the probe molecules, which are perfectly complementary to the probe molecules GC 67, GC 58 and GC 48 and which are each labeled with Cy3 at the 5 'end. It is 16 or 20mers (Fig. 4a, Fig. 4b).
  • the target nucleic acids have the properties shown in the table in FIG. 5.
  • the respective probe molecules are spotted in a concentration of 40 ⁇ mol on a spot diameter of about 120 ⁇ m on the above-mentioned glass slide from Schott.
  • Four identical microarrays are produced, which have measurement points in eight columns and three rows.
  • Negative control probes (GC 37) are spotted in the first two columns of each microarray, perfectmatch probe molecules PM are spotted in columns 3 and 4, and mismatch probe molecules MM1 in which a nucleotide has been exchanged are spotted in columns 5 and 6 , and in columns 7 and 8 mismatch probe molecules MM2 in which two nucleotides were exchanged were spotted.
  • the respective probe molecules PM, MM1 and MM2 are spotted with the melting temperature 67 ° C, in the second row those with the melting temperature 58 ° C, in column 3 those with the melting temperature 48 ° C.
  • This microarray thus has measuring points that cover a temperature range between 48 ° C and 67 ° C and thus of 19 ° C. 6 shows four such microarrays arranged one below the other.
  • a first microarray is mixed with a reaction mixture which contains all three of the 5'-Cy3 labeled oligonucleotides GC 67, GC 58 and GC 48 in a concentration of one nanomole per liter, at a hybridization temperature of 67 ° C. with the aid of an array booster Advalytix GmbH, Brunnthal, carried out with a mixing setting on the array booster of 26 dbm.
  • the signal intensities resulting from this experiment correspond to the signal intensities of the microarray surface shown first in FIG. 6.
  • a second microarray hybridization experiment at a hybridization temperature of 58 ° C. a result is obtained under otherwise identical conditions as it is schematically corresponds to the microarray shown in second place from above in FIG.
  • FIGS. 7A to 7D give the respectively measured signal intensities in the microarrays shown in FIG. 6 in the experiments carried out at 67 ° C. (FIG. 7A), 58 ° C. (FIG. 7B), 48 ° C (Fig. 7C), 38 ° C (Fig. 7D).
  • the result of the first hybridization experiment at 67 ° C. can be seen in FIG. 6 above.
  • the corresponding intensity values (detection of Cy3) are listed from FIG. 7A.
  • the mean value of the signal intensity at the measuring points that the GC 67 probe has is 28,507.25 units.
  • the signal intensity in the measuring point which has the GC 67 MM1 probe is only 1/100 of it, the signal intensity in the measuring points which contain the GC 67 MM2 probe molecules is even lower, it is less than a 1/300 of that in the Measuring point with the GC 67 probes. If the same experiment is carried out at a temperature of 58 ° C, the signal intensities determined at these measuring points are less far apart.
  • the signal intensity in the measurement point GC 67 MM2 is approximately 1/160 of the signal intensity determined in the measurement point GC 67, and the value determined in the measurement point GC 67 MM1 is approximately 1/7 of the value determined in the probe point GC 67 , Finally, at 48 ° C and 38 ° C, the factor shrinks to values between 1, 28 and 1, 88. At 58 ° C the factor formed from the signal intensities of the perfectmatch probes and the signal intensities of the respective mismatch probes is greatest for the GC 58 probes, whereas it decreases at the other temperatures for these probes.
  • the sequences of a second exemplary embodiment of the probes provided on the chip and of the corresponding target molecules are shown in FIG. 9.
  • the microarray of the second embodiment is basically the same as the microarray of the first embodiment, and differs from the first embodiment only in the other probe sequences.
  • This execution Example includes four PM test points for the melting temperatures 61 ° C, 53 ° C, 43 ° C and 33 ° C as well as corresponding MM 1 test points and MM2 test points.
  • a probe point is also provided for a negative control.
  • This microarray thus has measuring points that cover a temperature range between 33 ° C and 61 ° C and thus of 28 ° C.
  • the maximum temperature difference between two PM test points with neighboring melting temperatures is 10 ° C. This temperature difference should at least not be greater than 15 ° C. It can also be expedient to limit the maximum temperature difference between two PM test points with adjacent melting temperatures to less than 10 ° C., in particular to 8 ° C. or 5 ° C.
  • Cy-3 is used as a fluorescent marker in order to be able to determine the relative amount of hybrids formed in measuring points.
  • marking systems known to the person skilled in the art can also be used, for example different fluorescent dyes, or markings based on detection mechanisms other than fluorescence.
  • microarrays according to the invention can also have protein materials or other materials as probe molecules instead of nucleic acid material, depending on what type of microarray is to be examined with the system to be validated or calibrated.
  • the method according to the invention can be summarized as follows: a) Using a microarray with at least one first measuring point with probe molecules (PM) located therein, which are complementary to target molecules and which can form hybrids with the target molecules which have a melting temperature T m (PM) , - At least one second measuring point with probe molecules (MM1) located therein, which are incompletely complementary to the target molecules and which can form false hybrids with the target molecules, which have a melting temperature T m (MM1), b) Use of target molecules which form probe molecules (PM) are complementary in one or more first measuring points of the microarray, and c) performing a hybridization experiment with the microarray and the target molecules when a certain parameter is varied, d) determining the signal intensities as a function of the change in the parameter, the parameter value at the maximum difference between the signal intensities of the first and second measuring points being the parameter value at which the system approximately approximates the hybrids in the first measuring point is in balance and to which the system is calibrated.

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Abstract

L'invention concerne un procédé servant à calibrer et/ou à valider un système pour l'exécution d'une expérience à jeu ordonné de micro-échantillons, ainsi qu'un jeu ordonné de micro-échantillons et un nécessaire correspondants. Le procédé selon l'invention comprend les étapes suivantes : a) mise à disposition d'un jeu ordonné de micro-échantillons comprenant - au moins un premier point de mesure dans lequel se trouvent des molécules sondes (PM) qui sont complémentaires de molécules cibles et qui peuvent former avec les molécules cibles des hybrides ayant une température de fusion Tm(PM), - au moins un deuxième point de mesure dans lequel se trouvent des molécules sondes (MM1) qui sont partiellement complémentaires des molécules cibles et qui peuvent former avec les molécules cibles des hybrides partiels ayant une température de fusion Tm(MM1) ; b) mise à disposition de molécules cibles complémentaires de molécules sondes (PM) dans un ou plusieurs premiers points de mesure du jeu ordonné de micro-échantillons ; c) exécution d'une expérience d'hybridation avec le jeu ordonné de micro-échantillons et les molécules cibles en faisant varier un paramètre défini ; d) détermination des intensités de signal en fonction de la variation du paramètre, la valeur du paramètre à la différence maximale entre les intensités de signal des premiers et des deuxièmes points de mesure étant la valeur du paramètre pour laquelle le système est approximativement en équilibre eu égard aux hybrides dans le premier point de mesure et sur laquelle on calibre le système.
EP04804017A 2003-12-23 2004-12-17 Procede pour valider et/ou calibrer un systeme pour l'execution d'experiences d'hybridation, jeu ordonne de micro-echantillons et necessaire correspondants Withdrawn EP1706505A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003161137 DE10361137A1 (de) 2003-12-23 2003-12-23 Mikroarray, Kit und Verfahren zum Validieren und/oder Kalibrieren eines Systems von Hybridisierungsexperimenten
PCT/EP2004/014414 WO2005064012A2 (fr) 2003-12-23 2004-12-17 Procede pour valider et/ou calibrer un systeme pour l'execution d'experiences d'hybridation, jeu ordonne de micro-echantillons et necessaire correspondants

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DE102007055386B4 (de) 2007-11-20 2015-07-16 Boehringer Ingelheim Vetmedica Gmbh Verfahren zur Kalibrierung eines Sensorelements
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US6489096B1 (en) * 1998-10-15 2002-12-03 Princeton University Quantitative analysis of hybridization patterns and intensities in oligonucleotide arrays
US6136541A (en) * 1999-02-22 2000-10-24 Vialogy Corporation Method and apparatus for analyzing hybridized biochip patterns using resonance interactions employing quantum expressor functions
WO2001066804A2 (fr) * 2000-03-09 2001-09-13 Protogene Laboratories, Inc. Methodes permettant d'optimiser la capacite d'hybridation de sondes polynucleotidiques, et permettant de localiser et de detecter des variations de sequence
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AU2002252297A1 (en) * 2001-03-10 2002-09-24 Bioinformatics Dna Codes, Llc Methods and tools for nucleic acid sequence analysis selection and generation
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