WO2002018945A2

WO2002018945A2 - Analysis chip

Info

Publication number: WO2002018945A2
Application number: PCT/EP2001/009286
Authority: WO
Inventors: Ralph Müller
Original assignee: Basf-Lynx Bioscience Ag
Priority date: 2000-08-30
Filing date: 2001-08-10
Publication date: 2002-03-07
Also published as: DE10042797A1; WO2002018945A3; DE10042797C2; AU2002212136A1

Abstract

The invention relates to an analysis chip (10), e.g. a DNA array, on which a number of different types of molecules are immobilised in allocated spatial areas (14), respectively. A corresponding code is allocated to each type of molecule in a corresponding spatial area (24) on the chip. The code indicates which type of molecule is immobilised in the respective area. The code can be formulated according to the arrangement of the molecules, for example, if in an area of 5x5 spots, only certain spots are occupied, a binary code is formed. Alternatively, the code (24) can be formed on the chip independently of the immobilised molecules, e.g. as a two-dimensional binary code. The degree of certainty in the use of DNA arrays is increased since when the chips are read out with the help of the respective codes, it is even possible to determine which target molecules have been bound to and so which molecules were in the sample, without having recourse to external information.

Description

Analysis chip

description

The invention relates to an analysis chip on which different types of molecules are immobilized in the respectively assigned spatial areas.

In an increasing number of medical and scientific applications, samples containing DNA or RNA molecules are being analyzed qualitatively or quantitatively using suitable DNA arrays. During the analysis, the binding or hybridization of the DNA molecules obtained to suitable DNA fragments, so-called targets, immobilized on the array is detected.

This requires the production of suitable DNA arrays, which are also called biochips. These are usually produced from microtiter plates that contain solutions with suitable DNA fragments in the wells. The wells are small wells with a volume per well of, for example, 20 μl. Each well contains a known, specially synthesized DNA fragment. To build a DNA array, e.g. 220 pl solution from a well to a precisely defined position on e.g. pipetted on a slide. This is done by so-called spotting robots. The pipetted DNA fragments are then immobilized on the slide or chip or target carrier.

The sample to be analyzed is then placed on the target carrier or the DNA array. The sample contains radioactive or colored DNA or RNA molecules. Hybridization takes place in a special hybridization chamber at a suitable temperature. Non-hybridized or non-specifically bound DNA or RNA from the sample to be examined is removed by rinsing. hybridized DNA or RNA molecules are detected according to their labeling in a reader.

In principle, detection of radioactive radiation from the sample and detection of fluorescence signals can be considered. Fluorescence signals can result from fluorescence labeling of the sample or target or by intercalators. Energy transfer or mechanisms of fluorescence quenching between sample and target can also be used.

Difficulties arise in that such DNA arrays are constructed differently from manufacturer to manufacturer. Their exact structure generally depends on the respective filling of the microtiter plates and the arrangement of the applicators (needles, piezo nozzles, etc.) of the spotting robots. The different DNA array formats and designs easily lead to evaluation errors that arise due to confusion or due to incorrect or incorrectly adapted evaluation masks. Even if the preparation for the evaluation is correct, faulty analysis of control or control spots can lead to evaluation errors due to shifting the evaluation mask. These errors lead to a high security risk in medical gene diagnosis, which is a main area of application of the DNA chips.

The object of the invention is to increase the security of analysis and evaluation when using arrays and DNA chips. This object is achieved by the inventions according to the independent claims. Advantageous developments of the inventions are characterized in the subclaims.

According to the invention, an analysis chip is used on which different types of molecules are immobilized in the respectively assigned spatial areas. Typically, these are DNA arrays. The immobilized molecules are then, for example, gene segments or oligonucleotides that uniquely identify a gene. However, it can also be antibody, protein, allergen arrays etc. or non-peptide chemical substance libraries. The analysis chips are usually used to detect binding reactions. However, the detection of an enzymatic activity is also possible. The spatial area assigned to a type of molecule is usually a rectangle or a circle, as it arises in spotting processes. Through multiple spots of a target within an area However, the area can also be distributed linearly or according to a predetermined scheme over the entire analysis chip in order to compensate for or find irregularities in the hybridization and to generate a redundancy of the hybridization reaction which increases the reliability of the marker analysis.

According to the invention, an associated code is formed on the analysis chip in an associated spatial area for each type of molecule, the code indicating what type of molecules is immobilized in the respective area.

On the one hand, each type of molecule is assigned a spatial area in which these molecules are immobilized. On the other hand, a code is assigned to each type of molecule. Each code in turn has a spatial area on the analysis chip in which it is formed. This associated spatial area can be identical to the spatial area in which the molecules are immobilized. However, it can also be adjacent to this spatial area or assigned to the immobilization area according to a fixed scheme.

In the event of a detection reaction, the code can be read out at the same time and thus it can be determined which type of molecules the sample has bound to. It is then no longer necessary to provide array-specific information about the type and arrangement of the molecules on a supplementary sheet or in accompanying software. This prevents confusion between different types of molecules. This significantly increases safety in handling, evaluation and diagnosis.

In a preferred development of the invention, the code is formed by the arrangement of the molecules themselves in the associated spatial area. In this way, the reaction result and the code can be read out in just one step.

This can be done in a particularly simple manner in that each spatial area on the analysis chip assigned to a predetermined type of molecules is divided into a plurality of spots, and in that the code is a binary code which is generated by immobilizing molecules in some of these spots are and not in the remaining spots.

The binary code can be encoded, for example, in that an immobilized spot has a 1 and a free spot has a zero. speaks, or vice versa. A database, which is generally accessible on the Internet, explains how the individual codes are assigned to the immobilized types of molecules. As a side effect, redundancy of the hybridization reaction is obtained because there is more than one spot for each type of molecule. Such a type of redundancy can be referred to as "internal" redundancy. "External" redundancy is spoken of if more than one code is assigned to a molecular type, so that the code of the molecular type can still be recognized under certain circumstances if a spot does not provide a complete hybridization signal, for example due to technical shortcomings, although this was to be expected would. The codes can be optimized for external redundancy with regard to fault tolerance. In the simplest case, this is achieved in that more bits are used per code, ^• would be needed as a minimum, about 4 are then both the 4-bit code instead of 3. The type of molecule, and assigned all three bit codes, that result when a spot or bit cannot be recognized.

Alternatively, the code can be formed on the analysis chip independently of the immobilized molecules. The code can then be generated using conventional means, such as a laser recorder such as the CD burner principle or a high-resolution printer. For example, the code can be arranged on the underside of the analysis chip. In this case, for a detection reaction, the code on the underside of the analysis chip is first used in an area clearly assigned to the molecular spots, e.g. the same spatial position. The analysis chip is then turned over and the hybridization signal, for example a fluorescence signal or radioactive radiation, is read out. In the following, the examples focus on general fluorescence signals.

In the case of transparent supports, such as. Glass or plastic films, code and fluorescence can be read out simultaneously.

Furthermore, code and spots can be applied on the same carrier side, target spots and code being in the same spatial area and the target spot (s) being applied to the code area.

Finally, the code can be formed between the spots, for example in the gaps between circular spots. In such a case le only one spot needs to be set up for each type of molecule. The code can be applied using common technical means, such as laser marking or microspotting (piezo, pin, imprint, etc.).

The code is preferably designed in the form of a two-dimensional barcode. This can be based on current standards, such as the type C symbol from the .One code from Axtel, Inc., Fountain Valley, CA 92708, USA, www.Axtel.com, which can encode 64 alphanumeric characters.

The code could then encode an alphanumeric string that represents the name of the immobilized type of molecule, for example the annotation of the gene as defined in a publicly accessible database. It was then no longer necessary to explain in a database that is generally accessible on the Internet, how the individual codes are assigned to the immobilized types of molecules. Sufficiently would in such a case, the simple indication of Code One Type C von Axtel- on the chip, associated with ^'a reference to the database, see the the sequences of the genes with the corresponding annotation.

In general, a standardized array code could be stored for each global database entry (e.g. in the EMBL database), which allows the clear and reliable assignment of molecular types to all two-dimensional or multi-dimensional storage formats. In order to further increase the security of the reading, position marks can also be formed on the analysis chip. These can e.g. are formed by one of the areas with a plurality of spots, each spot carrying immobilized molecules, while in the areas with molecules which are used for the binding reaction, not all spots are occupied. Because on the latter a code is formed, which consists of occupied and unused spots.

For example, in a range of 5 5 spots, only 5 spots could be occupied as a code with immobilized molecules, ie only 5 bits could be "set". This would result in a total of (25 over 5) coding possibilities, that is 53130. The position marks, on the other hand, could be characterized by the fact that all 25 spots are occupied and additionally serve as a basic pattern for the evaluation algorithm. In general, as few position marks as possible should be applied overall, since this does not take up too much spotting area.

With the array chips common today, a large number of position marks are required for a reasonably reliable reading.

In contrast, a position mark already enables a precise spatial orientation of the analysis chip in a chip carrying a code according to the invention.

Furthermore, the codes can be selected in such a way that if the evaluation mask is shifted by one line or column, no more useful code is recognized. On the one hand, this increases security against incorrect reading in the event of displacements. On the other hand, it can also be recognized that there is a mask shift here.

It makes sense to structure the code hierarchically. For example, the code can consist of a first part that describes the organism from which a gene comes, while a second part of the code names the gene itself. In this way, analysis chips can be set up that each carry all of the genes of humans or all of the genes of the mouse or another organism. The hierarchical code can be from. consist of a first and a second part. The first part of the hierarchical code can be arranged, for example, in the position marks, while the second part is arranged in the respective regions of the immobilized molecules. The type of coding of the second part of the code in the respective areas and the type of coding of the first part of the code in the position marks should clearly differ from one another so that the position marks can still be recognized as such.

For example, 5 bits out of 5 x 5 = 25 could be set for coding the annotation of a gene, while 5 bits out of 5 x 5 = 25 were not set for the organism from which the gene originated, i.e. 20 bits are set. In this way, a universal architecture for DNA arrays for genetic testing can be built.

In a further development of the invention, a second type of molecules can additionally be immobilized within a spatial area which is assigned to a first type of molecules. For both types of molecules, the associated code can be tion of the molecules within one spatial region.

If, for example, an area of 10 x 10 slots (these are placeholders for spots) is formed, of which only 3 bits are set (corresponding (100 over 3) = 161700 coding options), 97 slots remain free. The remaining 97 slots can still be used to save space and thus make better use of the analysis chip. A binding reaction can then be read out, for example, via two differently colored DNA fragments in the sample. With such multiple occupancy of an area, it should be avoided that two types of molecules are immobilized on one and the same slot, since such a ^• double occupancy leads to difficulties with Reading out fluorescence signals can result. Can be avoided this transit an appropriate selection of the types of molecules or ^'the code. In the simplest case, one code lies in a spatial half of the area and the second code in the remaining part of the area. Overall, the types of molecules and codes can be selected such that more than two types of molecules can be immobilized in one area. One can speak here of a selection for a maximum packing density, which could be adopted by a sorting algorithm when producing an analysis chip.

Multiple occupancy of a single slot is possible if the different types of immobilized molecules are colored differently within a single spatial area. The different colors can then be recognized when the analysis chip is read out. If you add a marked sample, the hybridization on all spots belonging to a certain code gives you both the color of the target and the color of the bound sample. In general, any form of spectroscopic characteristic within a spot can be used for identification. Energy transfer or fluorescence quenching between target and sample is also conceivable.

A further possibility of occupying slots several times can be achieved by designing the spots belonging to a type of molecule with a predetermined spatial pattern. For example, the spot for the individual types of target molecules can be in the form of an arrow, egg or eccentric, ie one Object that has a preferred direction that is oriented differently depending on the type of molecule. Such patterns can be applied particularly easily when the target is transferred to the analysis chip by means of stamps which can also be rotated. Spatial patterns can also be obtained, for example, by spotting a small circle and a large circle partially overlapping. In this case too, the spots belonging to a certain code can be recognized by the fact that they have the same spatial pattern.

A combined use of color and shape to identify codes is also conceivable.

A further efficient use of the space within an area can be achieved in that the distance between the spots of a first type of molecule within the one area and the distance between the spots of a second type of molecule within the same area differ. For example, the distance from spot to spot (pitch) can be 100 μm for the first type of molecule and 98 μm for the second type of molecule, in the manner of a vernier. Such a shift is easy to see. In this way, a specific pitch can be assigned to each type of molecule.

Through a suitable evaluation of the readout signals, the spots of individual types of molecules can overlap, as can easily result from the different pitch. In general, the spot density can also be increased by a defined overlap of the spots.

The various possibilities of multiple use of an area mentioned above can all be combined with one another. The security in the use of analysis chips can also be increased in that after reading out a detection reaction on the analysis chip, the result of the detection reaction is written on the analysis chip. In this way, information, such as a finding for medical purposes, is retained even if, for example, the DNA on the chip should degrade. The analysis chip can then simply be given to the patient. The analysis chip can also carry one of the types of coding mentioned. The analysis result can be written to the analysis chip in a defined code form using customary means, for example by means of a laser, an ink jet or a laser printer. ckers, or other inscription-s _* procedure. The information should be readable as permanently as possible.

The invention is explained in more detail below with the aid of exemplary embodiments which are shown schematically in the figures.

The same reference numbers in the individual figures denote the same elements. In detail shows:

1A shows the schematic structure of a preferred DNA array;

1B shows an evaluation of the DNA array according to FIG. 1A;

2A shows the schematic structure of an alternative DNA array;

2B shows the schematic structure of a further alternative DNA array;

3 possibilities of spatial coding;

4 shows an area with spots of different pitch; and

Fig. 5 is a schematic representation of an analysis chip on which the result of a detection reaction can be written.

1A shows a DNA chip 10 with regions 12 arranged on a rectangular grid. Within each region 12 there is a grid of spots 14 on which oligonucleotides are immobilized. Position marks 16 are located at the outermost corners of the chip, which can be recognized by the fact that all spots 14 are occupied within their areas. Not all spots are occupied in one of the position marks 18, which can be recognized as a position mark due to its spatial position. Rather, this position marker 18 has a code for the word "human". The DNA chip 10 is therefore a chip for a genetic test on humans. The code in the position marker 18 can be built up, for example, by immobilizing dye molecules on individual spots in the simplest case, which corresponds to a set bit, while other spots are left free. In addition to dye molecules, double-stranded DNA molecules can also be immobilized, which are detected using intercalators.

In the center of the DNA chip 10 there is an area 20 which has a code for the annotation of a first gene, for example "actin", ie here oligo- or polynucleotides are immobilized which are clearly representative of the gene which is present in humans encodes the protein ACTIN. In a second area 22, a code for a second gene has been generated by immobilizing the associated oligo / polynucleotides. In this way, one gene can be encoded in each area of the DNA array. If the genes are ambiguous, further information such as splice and mutation variants, polymorphisms, sequence areas and lengths, etc. can also be encoded.

FIG. 1B shows the analysis chip according to FIG. 1A as it can be displayed on a screen after reading out by a fluorescence reader and evaluation. All areas 16, 18, 20, 22 are identified in their position and marked by rectangles. Areas 22 in which no hybridization signals could be recognized are crossed out. Areas 22 in which hybridization signals but no code could be recognized are e.g. marked with a crossed out full circle. Areas 20 in which a code was recognized are with the recognized code or the Em§orιS-Ät-2 <aήgtoes-telmr ÄiElmatischen structure of an alternative DNA array 10, in which the code is independent of the molecules immobilized in spots 14 is formed in areas 24 of the DNA array 10 which lie in the gaps between the essentially circular spots 14. Only one spot needs to be set for each type of molecule, which can also be relatively large. The DNA array 10 has a position marker 16 to increase the readout security. FIG. 2B shows a preferred embodiment of a variant of the DNA array according to FIG. 2A, in which the oligonucleotides are immobilized directly above the two-dimensional bar code. Since a thin layer of oligo / polynucleotides is essentially transparent, the code can also be read through the DNA or proteins without difficulty.

2A and 2B, the code is in the form of a two-dimensional bar code. In the preferred exemplary embodiment, the type A symbol from Code One from Axtel, Inc., Fountain Valley, CA 92708, USA, www.Axtel.com is used as the standard for the code. This standard allows the coding of 13 alphanumeric characters in 18 x 16 fields. Type C symbol, another variant, can encode 64 alphanumeric characters in 28 x 32 fields. Among other things, these codes are error-correcting, which further increases reading security. With a spot size in the code of approx. 5 μm, the code size of symbol type A of 18 x 16 spots and the resulting small size of the code areas of approx. 100 μm edge length, on a single DNA chip of approx. 10 cm ^Λ 2 size the entire human genome with its almost 100,000 genes can be made available for a test.

If Code One symbol type A is not used, but a simple binary code to designate the approximately 100,000 human genes, less than 20 bits are required. A range of 4x5 spots is therefore completely sufficient. To store the entire genome on a 10 cm ^A 2 chip, spot sizes of approx. 20 μm are sufficient.

If you don't want to store the entire genome but only selected genes, the spots can be significantly larger.

FIG. 3A shows a possibility of forming the spots belonging to a type of molecule with a predetermined spatial pattern, here by an arrow 26 for a first gene, an arrow 28 for a second gene, etc.

3B shows a further possibility of spatial coding. In this exemplary embodiment, two circles of essentially the same size are spotted side by side in different spatial arrangements, which results in spatial patterns 26 ', 28', etc. for different genes.

Fig. 3C shows the pattern of Fig. 3B when some of them are spotted on top of each other.

3D shows a further possibility of spatial coding. In this exemplary embodiment, eccentrics with different spatial orientations are spotted essentially overlapping, which results in spatial patterns 26 ″, 28 ″, etc. for different genes. The large circle in the middle contains immobilized molecules for all genes.

4 shows a further efficient use of the space within an area. The distance (pitch) XI of the spots 30 of a first type of molecule within the area and the distance X2 of the spots 32 of a second type of molecule within the area differ slightly in two dimensions. For example, the distance XI can be 100 μm and the distance X2 can be 98 μm, in the manner of a vernier. Such a shift is easy to see. A specific two-dimensional pitch can be assigned to each type of molecule. If only a few bits are set within a range, ie only a few spots are occupied for a code, the probability that two occupied spots overlap is relatively low. A gene sorting algorithm can guarantee the greatest possible packing density with the highest level of detection reliability.

FIG. 5 shows a DNA array 34 onto which the result of the detection reaction can be written after reading out a detection reaction. For this purpose, the DNA array 34 shows a section 36 in which the result can be written using conventional means. Furthermore, the DNA array 34 shows a section 38 in which target molecules are immobilized. Which molecules are immobilized in the individual regions 40 of section 38 is indicated by a code which is represented by the spatial immobilization pattern. How the code is to be read is specified in a third section 42 on the DNA array 34. In the present case, it is stated that Code Alpha is used. Code Alpha can be explained elsewhere, for example on the Internet. 5 corresponds to FIG. IB.

Numerous modifications and developments of the exemplary embodiments described can be implemented within the scope of the invention.

The code can be stored in an associated spatial area on the surface of the chip or inside the chip, for example in an electronic memory chip, which can be read out via a contact field, like a telephone card. In general, all storage media can be used as long as they are in or on the chip itself, e.g. also a magnetic stripe on the chip.

Results of a detection reaction can also be written to such an electronic or other memory on or in the chip. LIST OF REFERENCE NUMBERS

DNA chip area in which one type of molecule is immobilized in several spots spot position mark position mark in which the organism is coded area that has a two-dimensional bar code for the annotation of a first gene area that has a two-dimensional bar code for the annotation of a second gene Area for code that is independent of the immobilized molecules • Arrow ', 26' 'spatial' pattern Arrow ', 28' 'spatial pattern Spot of a first type of molecule - Distance between spots 30 of a first type of molecule Spot of a second type of molecule Distance between spots 32 a second type of molecule DNA array • Section in which the result of a detection reaction can be written Section in which target molecules are immobilized Area in which one type of molecule is immobilized in several spots Section that indicates the code used

Claims

claims

1. Analysis chip on which different types of molecules are immobilized in respectively assigned spatial areas (20, 22, 40), characterized in that for each type of molecules an associated code in an associated spatial area (20, 22, 24 ) is formed on the analysis chip, the code indicating what type of molecules is immobilized in the respective area.

2. Analysis chip according to claim 1, characterized in that the code is formed by the arrangement of the molecules in the associated spatial area (20, 22, 40).

3. Analysis chip according to claim 1 or 2, characterized in that each spatial region (20, 22, 40) assigned to a predetermined type of molecules on the analysis chip '(10, 34) into a plurality of spots (14) is divided; and - .. that the code is a binary code generated by the fact that molecules are immobilized in some of these spots and not in the remaining spots.

4. Analysis chip according to claim 3, characterized in that the code is designed such that it can still be recognized if the immobilization of the molecules in one of the spots cannot be recognized.

5. Analysis chip according to claim 1 or 2, characterized in that the code (24) is formed independently of the immobilized molecules on the analysis chip.

6. Analysis chip according to at least one of the preceding claims, characterized in that the code is in the form of a two-dimensional bar code.

7. Analysis chip according to at least one of the preceding claims, characterized in that the code encodes an alphanumeric string which represents the name of the immobilized type of molecules in each case.

8. Analysis chip according to at least one of the preceding claims, characterized in that additional position marks (16, 18) are formed on the analysis chip.

9. Analysis chip according to at least one of the preceding claims, characterized in that the code is structured hierarchically.

10. Analysis chip according to claim 8 and 9, characterized in that the hierarchical code consists of a first and a second part, - that the first part of the hierarchical code is arranged in at least one position mark (18); and that the second part of the hierarchical code is arranged in the respective regions (20, 22, 40) of the immobilized molecules.

11. Analysis chip according to at least one of claims 2, 3 and 6 to 10, characterized in that within a spatial area (20, 22, 40) which is assigned to a first type of molecules, a second type of molecules is additionally immobilized is; and that for both types of molecules the associated code is formed by the arrangement of the molecules within the one spatial area.

12. Analysis chip according to claim 11, characterized in that the types of molecules or the codes are selected such that both types of molecules are not immobilized on a common spot (14).

13. Analysis chip according to claim 11, characterized in that the different types of immobilized molecules within a single spatial area (20, 22, 40) are colored differently.

14. Analysis chip according to claim 11 or 13, characterized in that the spots (14) belonging to a type of molecule are formed with a predetermined spatial pattern (26, .26 ', 28, 28').

15. Analysis chip according to one of claims 11 to 14, characterized in that the distance (XI) of the spots (30) of the first type of molecule within the one area and the distance (X2) of the spots (32) of the second type of molecule within the Range differ.

16. Analysis chip on which different types of molecules are immobilized in the respectively assigned spatial areas, characterized in that after reading out a detection reaction on the analysis chip, the result of the detection reaction can be written on the analysis chip.

17. Procedure with the following steps:

A detection reaction is carried out by means of an analysis chip on which different types of molecules are immobilized in the respectively assigned spatial areas (40). After reading out the detection reaction, the result of the detection reaction is written onto the analysis chip.