DE10322282A1 - Automated genotyping, useful e.g. for assessing risk of developing thrombosis, comprises hybridization to a microarray of probes and evaluation against a control standard to indicate target sequences that can not be evaluated - Google Patents

Abstract

Method for automated genotyping, using a microarray of probes, in which the hybridization pattern is evaluated with reference to a control standard (CS) where failure to keep to CS indicates that the entire measurement, or individual target sequences being tested, can not be evaluated. Method for automated genotyping comprises: (a) hybridizing a sample containing target molecules (I) with a microarray that contains probes (II), complementary to wild-type and mutated sequences of (I); (b) reading the resulting hybridization pattern with a scanner; (c) using a computer-assisted unit for automated evaluation of the pattern, with reference to a control standard (CS), where failure to keep to CS indicates that the entire measurement, or individual target sequences being tested, can not be evaluated; and (d) automatic determination of the genotype of (I) from the hybridization intensities of (I) with both wild-type and mutant probes. An independent claim is also included for a system for performing the new method.

Description

The The invention relates to a method and a system for automatic Genotyping using a microarray.

A Many diseases such as cardiovascular diseases or the blood coagulation system are due to a variety of causes, taking not only the patient's lifestyle, but also whose genetic disposition is crucial. It therefore exists a need for genetic test systems that can carry out a genetic analysis of the Patients to determine their risk of disease reliably, quickly and inexpensive allow.

DNA microarrays basically allow implementation high throughput genetic analysis. In practice, however shown that a routine use of microarray technology in medical and diagnostic laboratories not easily possible is. This is because this technology is prone to failure and thus the establishment only with high technical and financial Effort is feasible. The handling of microarrays is also technical consuming. For this reason, trained personnel is necessary for an intended use enable in routine diagnostics.

in addition comes that the evaluation of the microarrays is also demanding and only be carried out by appropriately trained specialist personnel can. However, the evaluation is a crucial factor because of a misinterpretation of the results of misdiagnoses to lead can. The application of this technology in routine analysis was therefore impossible so far and was not considered to be possible in the future, since no procedure existed so far to the vulnerable and automate complex microarray systems so that it is largely without human verification reliable Results.

The The invention is therefore based on the object of a method and to provide a system that is largely automatic and reliable execution a DNA microarray analysis and its evaluation up to Genotyping enables.

Is solved this task by the method according to claim 1 and the system according to claim 14th

at the inventive method A microarray with probes is used for automatic genotyping to use that complementary to wild-type and mutation sequences of the individual to be examined Are target sequences. For example, on the microarray in addition to the wild-type sequences, the usual ones for the respective clinical picture are preferred relevant, functionally characterized mutation sequences applied, which is known or suspected to be present there is a genetic risk for the corresponding mutation. This for the selected microarray Sequences form the probe sequences.

The The sample to be examined is incubated with the microarray so that the target molecules present in the sample with the corresponding complementary probes hybridize. According to the invention the hybridized target molecules detected and the hybridization pattern of the microarray using of a reader read.

On An essential point of the present invention is the use of the computer-aided Evaluation unit which contains the data of the hybridization pattern read by means of the reading device based on control standards, particularly internal controls and include predetermined limit values, automatically evaluated. By the comparison of the read data with the specified limit values it is determined whether a certain characteristic or the whole test is evaluable. Thus, according to the invention, if the specified control standards are not complied with either the entire measurement was discarded or some of the examined Target sequences (features) based on the comparison with the specified ones Limit values declared as not evaluable. The affected to be examined Target sequence - how for example a certain gene mutation - is then preferably called not displayed so that the user knows that the target molecules the sample to be examined for this Target sequence could not be genotyped and this target sequence was therefore not recorded. By internal controls and predetermined Limit values as control standards ensure that only one Microarray or only such target sequences are evaluated and flow into the genotyping, where those are safe due to compliance with the specified limit values can be assumed that these can be evaluated and are reliable Deliver results. This overcomes the invention is a major problem in the prior art and allows a reliable Automation of the evaluation without the need for trained personnel is.

The genotype of the target molecules in the sample to be examined, for example the Pati enten is determined automatically on the basis of the determined hybridization intensity of the target molecules in the sample with the probes on the microarray. Due to the use of internal controls and specified limit values, only hybridization patterns that can be clearly determined are evaluated. The method according to the invention thus allows the routine use of the microarrays in diagnostics for the first time.

With the inventive method can advantageously known gene polymorphisms, for example, those with an elevated Risk of disease are associated in a single test approach routinely examined become. At the same time, the systematic investigation of combinations genetic risk factors without additional effort. Unlike with biochemical detection methods, due to the Genotyping is a distinction between heterozygous and homozygous feature media possible. The through the inventive method possible Creation of risk profiles and the detection of risk constellations help the treating doctor to clarify suspected diagnoses and necessary preventive measures initiate.

According to one advantageous further development of the present invention the results of the array automatically with an integrated database compared and comments corresponding to the respective results output. This makes the interpretation of the results significant facilitated. This advantageously turns into an expert system made available for genetic analysis using a microarray.

On Expert system is essentially a computer program, which Special knowledge from human experts in a limited area of responsibility reproduces and thus a problem with an expert comparable performance solves.

By the possibility the integrated database makes it easier for the attending physician to make the diagnosis.

The critical point regarding the functionality of such an expert system is available in the standing output data, since this is the basis for the evaluation by the Form expert system. Only if these are correct and reliable an expert system can be reliable work and thus output the correct result.

The inventive method allows the automatic determination of the genotype, due to of the planned internal controls and limit values will that only properly performed analyzes for Genotype determination can be used. This makes the routine use of an expert system is only possible and it makes it easier for the attending doctor to reduce the genetic risk to assess a patient. Can in the database For example, knowledge gained so far on already functional characterized mutations are included.

Preferably allows the database also makes statements about combinations of results. This involves commenting on results based on the presence are based on two or more mutations that are known to that they interact with each other. It is often so only combinations that make an illness probable, while the individual mutations for are less important on their own. By the inventive method and the creation of combination results is more advantageous Way the detection and evaluation of such mutations with synergy effects possible.

According to one advantageous further development of the method according to the invention a microreaction vessel is used, in which the microarray is integrated (see for example the German patent application 102 01 463.9 dated January 16, 2002 of ClonDiag Chip Technologies, Jena, “Reaction vessel for carrying out Array method "). The probe array is preferably at the bottom of the tube arranged. The use of such a combined microreaction vessel enables that all necessary work steps are carried out directly in a reaction vessel with common laboratory equipment can. This makes it cumbersome and handles that contain a potential for error can be avoided, so that the procedure is also carried out by personnel who have not been specially trained Briefing possible is. The use of a microreaction vessel is also included integrated microarray for the implementation hybridization is an advantage. In this way, the microreaction vessel ensures homogeneous Wetting and tempering the microarray for all reaction steps. Read this often caused by handling special hybridization chambers Avoid artifacts, what the quality of hybridization and thus that of the individual Array investigations improved.

The DNA microarrays are preferably provided with probes created for the respective clinical picture to be examined, which are either applied by standard immobilization processes or by a special in situ synthesis process (see for example DE 197 06 570 C1 ) be generated.

Preferably the individual probes are applied several times on the microarray, to enable multiple evaluations. This increases the quality of the results. According to the preferred embodiment the individual probes are preferably at least three times each four times, applied. Furthermore, it is advantageous to use the microarray Arrange sequences that act as internal controls. This Controls include, for example, those for checking the polymerase chain reaction, those to check the Hybridization process, as well as those for checking the silver color. Thereby external controls, which involve additional work, are largely unnecessary.

Various known technologies can be used to detect the hybridized molecules. The target sequences can be detected either directly or indirectly, for example with fluorescent dyes, with enzymes, by means of chemiluminescence, or with radioactivity. Physical methods such as mass spectrometry can also be used to detect hybridization reactions. According to the invention, a silver marking is preferably used for the detection of hybridized molecules (see, for example, WO 02/002810 "Method for the qualitative and / or quantitative detection of molecular interaction on probe arrays") This is in detail in 2 described. Depending on the chosen detection method, a suitable reading device is selected.

Especially the combination of the silver detection with a densitometric reading device is preferred. The silver color has across from fluorescence-based methods the advantage that fluctuations in results, by fading the for the sample label usually Fluorophores used occur in the inventive method according to this variant does not matter. The use of the reader enables the spatially resolved, sensitive Detection of the silver precipitate formed during the detection on the probe array. This will keep track of the time Course of the silver precipitation and thus the semi-quantitative Allows detection of target hybridization. Due to the temporal Tracking silver precipitation the dynamic measuring range and the sensitivity of the analysis can be expanded increase.

The inventive method basically takes place Use For Genotype and mutation analyzes.

A preferred application for the inventive method represents the analysis of the innate tendency to thrombosis (thrombophilia). About two out of a thousand people in Germany are in the course of their Life affected by deep leg or pelvic vein thrombosis. Die annually in Germany up to 40,000 patients with the most serious complication deep vein thrombosis, pulmonary embolism. In addition to risk factors like smoking, obesity, temporary immobilization, pregnancy and oral contraceptive use thrombophilia is largely genetic. More than half of the Thrombosis patients are genetically predisposed.

To the most common changes In the genetic material, which increase the risk of thrombosis, mutations in different count Genes for the coagulation factors. Here in particular are the point mutations Factor V Leiden and Factor II G20210A (Factor II) should be mentioned. In Recently, various polymorphisms in genes of the Homocysteine metabolism (MTHFR, CBS, MTR) with an increased risk of thrombosis connected. By combining the mutations and corresponding synergy effects can increase the risk significantly again. Other factors, such as smoking, can be added the risk of thrombosis increases again many times over.

For example have heterozygous traits for factor V suffering five times, homozygous carriers even a fifty- up to a hundred times higher Risk of thrombosis. While carrier of the heterozygous defect of factor II a threefold increased risk of thrombosis have, increases the simultaneous intake of oral contraceptives the risk a hundred and fifty times.

• – Faktor V Leiden
• – Faktor V Cambridge
• – Faktor II G20210A
• – MTHFR C677T
• – MTHFR A1298C
• – CBS 844 ins 68
• – PAI-14G/5G
• – MTR A2756G.

A microarray for detecting the risk of thrombophilia preferably has the probes for detecting the following polymorphisms:

• - Factor V suffering
• - V Cambridge factor
• - Factor II G20210A
• - MTHFR C677T
• - MTHFR A1298C
• - CBS 844 into 68
• - PAI-14G / 5G
• - MTR A2756G.

This Polymorphisms form in the case of such a thrombophilia microarray the preferred target sequences to be examined. The genotype of the target molecules in a sample using such a microarray for this Target sequences are determined.

• – ApoB Arg3500Gln
• – ApoB Arg3531 Cys
• – ApoE E2, E3, E4
• – MTHFR Ala222Val
• – PAI-1 4G/5G
• – LPL Asn291Ser
• – ACE 287 bp I/D in Intron 16
• – Angiotensinogen Met235Thr
• – CETP B1/B2
• – Cyp 7A1 – 204A→C
• – Paraoxonase 1 Gln192Arg
• – Glycoprotein III PLA1/A2.

A microarray for determining the risk of arteriosclerosis preferably has probes for detecting the following polymorphisms:

• - ApoB Arg3500Gln
• - ApoB Arg3531 Cys
• - ApoE E2, E3, E4
• - MTHFR Ala222Val
• - PAI-1 4G / 5G
• - LPL Asn291Ser
• - ACE 287 bp I / D in intron 16
• - Angiotensinogen Met235Thr
• - CETP B1 / B2
• - Cyp 7A1 - 204A → C
• - Paraoxonase 1 Gln192Arg
• - Glycoprotein III PLA1 / A2.

According to one advantageous further development of the method according to the invention are preferred internal controls to review the individual process steps carried out. Central sources of error are, for example, the success of silver staining, compliance with the hybridization conditions as well as the PCR for sample preparation. Through the controls guaranteed that a microarray is only evaluated and as the basis for a diagnosis serves if the procedure was carried out in the normal range.

With The invention also provides an automatic genotyping system to disposal posed.

This has a microreaction vessel with a integrated microarray. The microarray carries oligonucleotides that are complementary to selected wild-type and mutation sequences of target sequences to be diagnosed are. The advantage of using a microreaction vessel with integrated microarray has already been explained. The test system also includes a detection reagent for the detection of hybridized target molecules. To the The silver coloration explained above is preferably used for detection. The system according to the invention also has a preferably densitometric reader for reading of the hybridization pattern on the microarray.

core element of the system according to the invention is a computer based Evaluation unit for automatic evaluation of the read data from the Hybridization pattern based on specified control standards (such as internal controls and specified limit values in particular) and for the automatic determination of the genotype of the diagnosed Target sequences. If the specified limit values are not observed either the entire measurement or individual due to the Comparison with the limit values classified as not evaluable Target sequences to be diagnosed declared not evaluable. Thereby is in turn guaranteed that only such a microarray or only such target sequences be evaluated and included in the genotyping where can be safely said due to compliance with specified limit values can be evaluable and deliver reliable results. The determination of the genotype is based on the hybridization intensity of the target molecules in the Sample determined with the wild-type and mutation probes of the microarray. The advantage of such an evaluation unit was related already explained in detail with the method.

The The system preferably also has one connected to the evaluation unit Database on which comments on the identified genotypes of the has investigating target sequences. The advantages of such integrated database have already been explained in detail.

With available system, the method according to the invention can be carried out and consequently has the associated advantages already described on.

The Invention will now be explained in detail with reference to figures:

1 shows a microreaction vessel with a permanently integrated microarray and sample images for hybridized microarrays,

2 is a schematic representation of the silver label for the detection of the specifically hybridized target molecules,

3 4 is a schematic flow diagram for array evaluation according to the preferred embodiment;

4 is a diagram for evaluating the hybridization pattern for examining the risk factors of thrombophilia,

5 shows examples of results comments.

1 shows the microreaction vessel that is preferably used 1 , This is a standard micro-reaction vessel with a microarray firmly integrated at the bottom of the tube 2 , A synthesized array is shown enlarged 3 , which according to exemplary embodiment has 4096 different oligonucleotides, each of which is twenty-four bases long and forms spots with a size of 32 μm in diameter. The total array area is 2048 × 2048 μm. With 4 is marked a spotted array that has 120 spotted oligonucleotides.

2 explains the preferably used Silver marking for the detection of the specifically hybridized target molecules. On the carrier 5 is a probe 6 immobilized. In hybridization, double strands form between complementary partners. With 7 such a hybridized target molecule or the double helix of probe and target molecule is identified. The target molecule carries a biotin label 8th , The specifically hybridized target molecules are provided with gold particles. This is done using a streptavidin-gold conjugate 9 which binds to the biotin label. Then an amplifier solution containing silver ions is added. A silver precipitate forms 10 on the streptavidin-gold-biotin complex, which depicts the specific hybridization pattern of the investigated sample. The silver precipitate on the probe array is recorded in a densitometric reader.

3 explains in a flowchart preferred steps for evaluating the hybridization pattern read. In step 1 it is first checked whether discrete spots can be recognized on the array. In step 2 The intensity of the background over the entire array is determined during the evaluation. This is done without considering the probes. For example, the background can be determined by averaging four points at four corners of the array and one point in the middle of the array. It then takes place in the crotch 3 a comparison of the measured intensity of the background with an internal control, which preferably consists of an oligonucleotide labeled with biotin. If the comparison shows that the background is low-signal and homogeneous, the evaluation is continued. If this is not the case, however, the evaluation is terminated and the array is classified as non-evaluable. The permissible deviation of the signal intensity of the background is preferably less than or equal to 30%. For the comparison, the quotient is in each case formed from the intensity of the measured background and the intensity of the oligonucleotide labeled with biotin, and the values are compared with one another. In addition, the signal intensity of the background should preferably not exceed a predetermined ratio to the signal intensity of the control probe.

The steps 4 . 5 and 6 are preferred controls by which it can be determined whether the procedure has been carried out correctly and thus provides reliable results.

So in step 4 a control with regard to the silver staining was carried out in such a way that an oligonucleotide labeled with biotin is detected directly by the gold / silver labeling by binding gold-labeled streptavidin. This probe can also be used in step 3 described background intensity check can be used. In step 5 the hybridization process is controlled by hybridizing a probe to a synthetic target labeled with biotin. This target is part of the hybridization solution, which also contains the target molecules of the patient sample. The hybridization of this synthetic target is checked via the intensity of the silver coloration. If the specified limits for the internal controls in step are not met 4 and step 5 the entire microarray is automatically declared not evaluable and a corresponding error message appears.

In step 6 the functionality of the polymerase chain reaction is checked. The check can be carried out for each individual target sequence to be examined. This is done, for example, by the fact that there are probes on the array that are complementary to highly conserved sections of the target molecules. If the patient sample is successfully amplified, hybridization occurs regardless of the patient's genotype. If such a hybridization is not detected, it can be seen that the amplification of the respective target molecule in the patient sample was faulty and the corresponding target sequence to be diagnosed is automatically excluded from the evaluation.

In step 7 the intensity of the individual spots is measured for each target sequence to be examined. This takes place both for the mutated sequence and for the corresponding wild-type sequence. At least three spots are measured for both the wild type and the mutated sequence in order to ensure additional internal control. Four spots are preferably measured.

In step 8th it is checked whether the measured values are homogeneous. As stated, triplets or combinations of four are preferably measured. Homogeneity is when the intensity of at least three spots deviates less than 30% from each other. If it is classified as homogeneous, step 9 the median of the intensity of the measured spots. In the event of inhomogeneity, the respective target sequence is declared not evaluable. In this case, an error message may appear that the respective target sequence X cannot be evaluated. For example, "Factor V cannot be evaluated" may appear on a display and / or in the result log. In this case it is clear that this characteristic (target sequence) could not be genotyped in the sample and is not recorded in the overall result.

If necessary, it can also be subsequently checked whether the median of the spot intensity exceeds a predetermined minimum value. When measuring triplets, at least one of the tri plets reach this minimum value. This can be 1.5 times the background, for example. If this is not the case and the measured intensity is too weak, the feature according to this variant can be declared as not evaluable by means of a corresponding error message.

In step 10 the quotient of median intensity wild type / median intensity mutant is formed for each characteristic or for each corresponding target sequence. The respective genotype can then be determined from this.

• – Sofern der Quotient aus Median-Intensität Wildtyp/Median-Intensität Mutant > 2,50 ist, gilt der Genotyp als Wildtyp, homozygot (Schritt 11),
• – Sofern der Quotient aus Median-Intensität Wildtyp/Median-Intensität Mutant 0,75 bis 1,5 beträgt, gilt der Genotyp als heterozygot (Schritt 12),
• – Sofern der Quotient aus Median-Intensität Wildtyp/Median-Intensität Mutant < 0,25 ist, gilt der Genotyp als Mutant, homozygot (Schritt 13).

Limit values are preferably set for determining the genotype for each target sequence to be diagnosed. During the development of the array, the limit values must be determined and determined experimentally for each target sequence to be examined. The following are examples of corresponding limit values:

• - If the quotient of median intensity wild type / median intensity mutant> 2.50, the genotype is considered wild type, homozygous (step 11 )
• - If the quotient of median intensity wild type / median intensity mutant is 0.75 to 1.5, the genotype is considered heterozygous (step 12 )
• - If the quotient of median intensity wild type / median intensity mutant <0.25, the genotype is considered to be mutant, homozygous (step 13 ).

Also a control step is preferably installed here. This defines that with a quotient of median intensity wild type / median intensity mutant between the specified limit values (according to the example from 0.25 to 0.74 or from 1.51 to 2.49) the corresponding target sequence / feature for not Explained Explainable becomes.

The Evaluation based on specified limit values and thus the determination of the genotype within the given parameters results in more advantageous Way to reliable Results so that misinterpretations are avoided.

The results are in step 14 collected and prepared for issue. For example, only the genetic variants or the heterozygous target sequences can be specified. Alternatively, all checked features can be output. In order to facilitate the interpretation by the examining doctor, the step 14 collected results preferably compared with an integrated database. Doing so in step 15 preferably first checked whether there is a comment specifically on the determined result constellation, for example in order to determine synergy effects or special risks in the presence of certain mutation constellations. In addition, each result comment in step 16 pulled from the database and in step 17 compiled. This can be done in the form of a table, for example. In step 18 the determined overall result is printed out.

4 shows the evaluation of a hybridization pattern in a diagram. This enables the genotype to be clearly identified.

5 shows sample texts for results of a thrombophilia chip. First, a text for a combination result is shown, which shows possible risks. Second, an individual result for the genotype determined is shown. These individual texts are preferably output even if there are result constellations.

Claims (23)

Method for automatic genotyping, characterized in that - a sample comprising target molecules is hybridized with a microarray which has probes which are complementary to wild-type and mutation sequences of target sequences to be examined, - the hybridization pattern of the microarray is read using a reader, - in the computerized evaluation unit automatically evaluates the hybridization pattern on the basis of control standards, - whereby if the specified control standards are not complied with, either the entire measurement or individual target sequences to be examined are displayed as not evaluable, - the genotype of the target molecules in the sample based on the hybridization intensity of the target molecules in the Sample is automatically determined with the wild type and mutation probes of the microarray. A method according to claim 1, characterized in that the results of the array are automatically integrated Database matched and comments corresponding to the results output become. A method according to claim 2, characterized in that a database is used that combines results comments having. Method according to one or more of claims 1 to 3, characterized in that the genotype of the investigated Target sequences by determining the quotient of intensity wild type / intensity mutant is determined. Method according to one or more of claims 1 to 4, characterized in that during the evaluation a) an automatic spot detection takes place, b) the intensity of the background of the microarray is determined, c) the background measured in b) is compared with an internal control, d) the intensity of the spots, at least three for wild type and mutation, are measured and compared with one another for the target sequences, e) the median of the measured intensities for wild type and mutant is formed, f) the quotient of median intensity wild-type / median intensity mutant is formed for each target sequence to be examined, g) the quotient from f) is used to determine the genotype of each target sequence to be examined. A method according to claim 5, characterized in that the allowable Deviation in step c) and / or d) = 30%. Method according to one or more of claims 4 to 6, characterized in that for the determination of the genotype of the target sequences to be examined are based on defined limit values such as: a) Is the quotient off Median intensity Wild-type / median intensity Mutant> 2.50 the wild-type genotype, homozygous; b) Is the quotient of median intensity wild type / median intensity mutant 0.75 to 1.5, the genotype is heterozygous; c) Is the quotient from median intensity Wild-type / median intensity Mutant <0.25, is the genotype mutant homozygous. Method according to one or more of claims 4 to 7, characterized in that at predetermined quotients from median intensity wild type / median intensity mutant from, for example, 0.26 to 1.49 or from 1.51 to 2.49 the corresponding target sequence is declared not evaluable. Method according to one or more of claims 1 to 8, characterized in that for the detection of hybridized molecules Silver mark is used. Method according to one or more of claims 1 to 9, characterized in that a microreaction vessel with a integrated microarray is used. Method according to one or more of claims 1 to 10, characterized in that a microarray is used, of probes for demonstrates the following polymorphisms: - factor V suffering - factor V Cambridge - factor II G20210A - MTHFR C677T - MTHFR A1298C - CBS 844 into 68 - PAI-1 4G / 5G - MTR A2756G and or - ApoB Arg3500Gln - ApoB Arg3531 Cys - ApoE E2, E3, E4 - MTHFR Ala222Val - PAI-1 4G / 5G - LPL Asn291 Ser - ACE 287 bp I / D in intron 16 - angiotensinogen Met235Thr - CETP B1 / B2 - Cyp 7A1 - 204A → C - Paraoxonase 1 Gln192Arg - glycoprotein III PLA1 / A2. Method according to one or more of claims 1 to 11, characterized in that a densitometric reader is used becomes. Method according to one or more of claims 1 to 12, characterized in that internal controls to verify Process steps, such as silver coloring, Hybridization conditions and PCR are carried out. Automatic genotyping system, comprising: - a microreaction vessel with a integrated microarray, which has oligonucleotide probes that are complementary to wild-type and mutation sequences of target sequences to be examined, - a device for reading the hybridization pattern on the microarray, - a computerized unit for automatic evaluation of the read data of the hybridization pattern based on specified control standards and for automatic Determination of the genotype of the target sequences to be examined, - in which if the specified control standards are not met either the entire measurement or individual target sequences to be examined are shown as not evaluable and - The genotype of the target molecules in the Sample based on the hybridization intensity of the target molecules in the Sample with the wild type and mutation probes of the microarray automatically is determined. System according to claim 14, characterized by a database connected to the evaluation unit, the comments on the determined genotypes of the target sequences to be examined contains. System according to claim 14 or 15, characterized through a densitometric reader. System according to one of claims 14 to 16, characterized by a microarray, the probes for the detection of the following polymorphisms: - Factor V Leiden - Factor V Cambridge - Factor II G20210A - MTHFR C677T - MTHFR A1298C - CBS 844 ins 68 - PAI-1 4G / 5G - MTR A2756G. and / or - ApoB Arg3500Gln - ApoB Arg3531 Cys - ApoE E2, E3, E4 - MTHFR Ala222Val - PAI-1 4G / 5G - LPL Asn291 Ser - ACE 287 bp I / D in Intron 16 - Angiotensinogen Met235Thr - CETP B1 / B2 - Cyp 7A1 - 204A? C - Paraoxonase 1 Gln192Arg - Glycoprotein III PLA1 / A2. System according to one or more of claims 14 to 17, characterized by an evaluation unit that at least the subsequent steps to determine the genotype of the subject to be examined Executes target sequences: a) execution automatic spot detection, b) Determination of the intensity of the background of the microarray, c) comparison of the background measured in b) with an internal control, d) measurement and comparison of the intensity of spots for everyone Target sequence to be examined, at least three each for wild type and mutation, e) Formation of the median from the measured intensities for wild type and mutant, f) Formation of the quotient from median intensity wild type / median intensity mutant for every target sequence to be examined, g) Using the quotient from f) to determine the genotype of each target sequence to be examined. System according to one or more of claims 15 to 17, characterized by an evaluation unit, h) those for the individual target sequences to be examined determined genotypes with a Database compares, giving an output of the respective results assigned comments of the database. System according to one or more of claims 17 to 19, characterized by an evaluation unit for step c.) and / or d.) a permissible Based on a deviation of 30%. System according to one or more of claims 17 to 20, characterized by an evaluation unit for the determination of the genotype of the target sequences to be examined Based on limit values, for example: a) Is the quotient from median intensity Wild-type / median intensity Mutant> 2.50 the genotype homozygous, wild type; b) Is the quotient of median intensity wild type / median intensity mutant 0.75 to 1.5, the genotype is heterozygous; c) Is the quotient from median intensity Wild-type / median intensity Mutant <0.25, is the genotype homozygous, mutant. System according to one or more of claims 17 to 21, characterized in that at predetermined quotients from median intensity wild type / median intensity mutant from, for example, 0.26 to 1.49 or from 1.51 to 2.49 the corresponding target sequence is declared not evaluable. System according to one or more of claims 15 to 22, characterized by a database, the combined results comments contains.