WO2003048385A2

WO2003048385A2 - Oligonucleotide array, nucleotide detection method and device therefor

Info

Publication number: WO2003048385A2
Application number: PCT/EP2002/013861
Authority: WO
Inventors: Stefanie WASCHÜTZA; Alf-Andreas Krehan; Oliver BÖCHER
Original assignee: Adnagen Ag
Priority date: 2001-12-06
Filing date: 2002-12-06
Publication date: 2003-06-12
Also published as: DE10159904A1; WO2003048385A3; AU2002349054A1; EP1456406A2

Abstract

The invention relates to an oligonucleotide array comprising a substrate encompassing at least one field, wherein oligonucleotides with a field-specific sequence are immobilized in said at least one field. The immobilized oligonucleotides of the at least one field are modified in such a way that they are stable as a single strand and/or double strand with respect to enzymatic, chemical, physical and/or chemical-physical degradation.

Description

Oligonucleotide arrangement, method for nucleotide detection and device therefor

The present invention relates to an oligonucleotide arrangement, for example an oligonucleotide array such as the DNA chips and the like. the like , a method for the detection of oligonucleotides and an apparatus for carrying out such methods. Such oligonucleotide arrangements are used to simultaneously detect or check a large number of oligonucleotide sequences by means of their individual fields. The respective fields of the nucleotide arrays contain oligonucleotides in immobilized form, which have a complementary sequence to the oligonucleotides to be detected. If there is now a corresponding oligonucleotide in a sample, it is hybridized to the corresponding complementary oligonucleotide in a specific field of the array and can then be detected there. However, the direct hybridization of arrays with genomic DNA generally does not achieve the required sensitivity of the signal subsequently detected due to a complementary binding of nucleic acids. Therefore, a selective duplication (e.g. by PCR) of individual sequences upstream of the hybridization is generally required. This must be established separately for each array and has a quantitative limitation (for example as a multiplex, in which no more than 50 different sequences can be amplified at the same time, although the amplification efficiency for individual sequences can also differ with this complexity.

Another difficulty is the non-specific binding, which leads to disturbing background signals. This problem exists in hybridization with amplified oligonucleotides as well as in hybridization with genomic DNA.

Another difficulty is that in order to obtain a detectable signal, the binding component either has to be provided directly with a fluorescent dye or has to have a chemical modification which enables subsequent radioactive or fluorescent labeling. In the first case there are handling restrictions for the hybridized array, since it must be kept away from light, in the second case at least one further marking and washing step is required, which extends the overall procedure and thus makes it less efficient.

The object of the present invention is now a

Oligonucleotide arrangement, for example a DNA chip, to provide, with which the hybridization of oligonucleotides in the individual fields can be detected in a simple, safe and highly sensitive manner. Furthermore, it is an object to provide a corresponding detection method and a device for this.

This object is achieved by the oligonucleotide arrangement according to claim 1, the method according to claim 7 and the device according to claim 32. Advantageous further developments of the oligonucleotide arrangement according to the invention, the method according to the invention and the device according to the invention are given in the respective claims.

With the method according to the invention, nucleotide sequences are now detected by binding them to complementary oligonucleotides immobilized on a substrate. Then there is a breakdown, for example a digestion, for example a restriction digest, with which essentially or specifically single-stranded nucleic acids are broken down. Chemical, physical or chemical-physical degradation is also possible. Double-stranded nucleic acids thus remain on the substrate and can then be detected. This can be done, for example, by the fact that the actual nucleotide sequences to be detected are already marked and so only the markers immobilized on the substrate are to be detected. On the other hand, double-stranded nucleic acids can also be detected, for example, by intercalating dyes. The degradation of the single-stranded DNA on the substrate causes a reduction in the background. The nucleotide sequences to be detected can be, for example, nucleic acids plotted by PCR or directly non-amplified genomic DNA or RNA can also be used. The degradation can take place chemically or also by means of suitable restriction endonucleases, for example the S1 nuclease.

Are probe sequences immobilized on the substrate, which are protected against the respective degradation as a single strand and / or as a double strand, i.e. if they are stable, repeated hybridization with the nucleotide sequences to be detected can take place, the hybridization being followed in each case by a corresponding degradation or digestion step. As a result, hybridized nucleotide sequences to be detected can be enriched on the chip over time, so that the sensitivity of the detection method increases considerably.

Suitable degradation processes are, for example, the degradation of nucleic acid by means of chemicals, for example by means of hydroxylamine and osmium tetroxide, followed by cleavage with piperidine or the degradation by hydrazine with piperidine or else the degradation by dimethyl sulfoxide (DMS), formic acid and piperidine. Alternative degradation methods use restriction endonucleases, for example SI nucleases. Also a breakdown by

DNA cleavage using radiation can be used.

An example of such an oligonucleotide arrangement and its use in the method according to the invention are described below.

Sequences are applied to an array which contain thioesters instead of a phosphoric acid ester bond in the sugar-phosphate backbone of the oligonucleotides (thiophosphonates). The hybridization takes place with fragmented genomic DNA without prior amplification at the optimal hybridization temperature for the corresponding sequences. In order to achieve complete and specific hybridization, this hybridization step is repeated cyclically with a new application of fragmented, unamplified DNA. The DNA can be fragmented, for example, by at least partial restriction digestion or by shearing the DNA. Only small amounts of genomic DNA are used to further increase the specificity (instead of about 10 ng / 30 μl with an area of 4 qcm, this is reduced to approx. 1 ng). Subsequent to each hybridization step, the hybridization solution is replaced by a solution for restriction digestion with S1 nuclease, the temperature being reduced to 37 ° C. The Sl nuclease M5761 from Promega was used here as an example of the Sl nuclease. In this way, single strands of all types, such as those that occur in the case of incorrect hybridizations, and protruding strand ends are eliminated. A further increase in temperature to the hybridization temperature denatures the restriction enzyme and thus prevents the digestion of single-stranded DNA for the next following hybridization step. In addition, remaining short DNA sections, which, however, do not hybridize with the entire oligonucleotide, are detached without detaching completely hybridized DNA sections to be detected. Then DNA solution (genomic, non-amplified, sheared DNA without modifications) is applied again. This cyclically repeated hybridization can be simplified by automatically changing the solutions. In this way, complete saturation of all oligonucleotides is achieved at one application site, while incomplete and incorrect hybridizations are avoided become; in sequences without a genomic counterpart there is no double strand after hybridization.

A complete background reduction is possible due to the fact that the nucleases degrade or cut the incompletely hybridized oligonucleotides from the sample and the protruding nucleotides of the strand end of a fully hybridized oligonucleotide, which are each present as a single strand. Because the remaining fragments, which continue to bind to the immobilized oligonucleotides after the single-stranded nucleotide regions have been broken down, detach even with a slight heating, so that the corresponding oligonucleotides, which are immobilized on the fields, then again completely for the next cycle for hybridization. reaction are available. So it is possible to remove any background.

After complete hybridization, an optical differentiation between hybridized and non-hybridized sequences is necessary. This can be done in different ways. One possibility is to use a fluorescent dye intercalating in double-stranded DNA, which also enables absolute quantification. For this purpose, after the last hybridization / SI digestion, the temperature is brought to a value which is slightly below the hybridization temperature and a solution with a fluorescent dye which selectively intercalates in double-stranded DNA is added. This enables highly specific coloring without a background to take place in a relatively short time. An absolute quantification is then possible through the proportional intercalation of the fluorescent dye. The following methods are also suitable as detection methods for the ultimately double-stranded nucleic acid of each individual field:

• Use of fluorescent dyes intercalating in DNA double strands;

• Use of substances intercalating in DNA double strands with modification (e.g. biotin) via which a fluorescent or other dye can be bound;

• Use of substances intercalating in DNA double strands with modification (e.g. biotin), via which an enzyme can be bound which catalyzes a color reaction;

• Use of substances intercalating in DNA double strands with modification (e.g. biotin), via which an enzyme can be bound which catalyzes a bio- or chemiluminescence reaction;

• Use of substances intercalating in DNA double strands with modification (e.g. biotin), via which a radioactively labeled component can be specifically bound;

• Use of a component which, in a redox reaction, adds covalently to the marginal sugar of the binding strand and can be identified as a dye / fluorescent dye / radioactive label or a Che i or bioluminescence reaction. Furthermore, it is possible to match the individual hybridization temperatures of the individual array fields to one another in order to achieve an approximately uniform hybridization. This can be achieved, for example, by chemical modification of the immobilized oligonucleotides, for example on the ribose component, or by a suitable choice of the oligonucleotide length for each individual oligonucleotide field.

Overall, the method according to the invention has the following advantages:

Absolute quantification is possible, the LNA technology for point mutations can be avoided, there is still no separate marking step with appropriate process management and detection using intercalating substances, the entire process can be automated and enables the use of genomic DNA without any chemical modification or without any previous reproduction. This means that the sample preparation takes little time and money. There is maximum flexibility with regard to the marking technology, since a wide variety of markers can be used.

In summary, the following special features distinguish the technology according to the invention from the prior art:

A selective step of breaking down, for example restriction cleavage on the array, is carried out;

# by breaking down or digesting protruding ends of the hybridized nucleotides from the sample these are removed so that intercalation dyes can be used without creating an additional background;

• by breaking down or digesting single-stranded sections (protruding ends and incompletely hybridized oligonucleotides), unspecific background is removed;

Stabilized immobilized oligonucleotides are advantageously used to enable repeated hybridizations;

• The essential property of the immobilized oligonucleotides is their resistance to the

Degradation of single-stranded DNA sections, for example against digestion by single-strand cleaving nucleases;

• additionally modified oligonucleotides have a higher binding intensity. This enables a higher, more easily compensated hybridization temperature;

• Restriction enzyme used is inactivated by increasing the temperature, which is necessary for the next cycle anyway;

• a repeated repetition of the process until saturation is possible;

• The intercalation dye can only be added at the end of the process, thereby avoiding light-protected handling during the previous hybridizations. In the following, 4 figures show a device according to the invention and the basic process sequence.

Show it:

Figure 1 shows a device according to the invention in cross section.

2 shows a top view of a device according to the invention;

Fig. 3 is a plan view of a cross section through an inventive device and

Fig. 4 shows the basic process flow of the method according to the invention.

In FIG. 1, reference number 1 denotes an apparatus according to the invention for carrying out the detection method according to the invention. This has a carrier 2, which has a recess 3 as a hybridization chamber. This hybridization chamber 3 is closed with a cover 6 which rests on the carrier 2. The carrier 2 also contains a heating element in order to heat or cool the hybridization chamber. However, the heating element can also be located below / outside the carrier in the surrounding device. The heating or cooling can be carried out, for example, by a Peltier element. The hybridization chamber 3 has a further recess with a narrower cross section, which is designed as an oligonucleotide array according to the invention.

2 also shows a device according to the invention, the same or similar elements having the same or similar reference numerals as in the following. te are designated. A hybridization chamber 3, which is designed as a DNS chip, is also introduced into a carrier 2. Via feeds 6, 7, 8 and 9, for example, sample liquids or washing solutions or nuclease solutions can be passed over the DNA chip and can be derived again via a drain 10. Inlets 6 to 9 can be controlled, for example, via magnetically controlled valves and a corresponding microprocessor control.

FIG. 3 again shows a device 1 with a carrier 2, a hybridization chamber 3 and a heating and cooling element 4, with which the hybridization chamber 3 and thus the DNS chip can be heated or cooled. In principle, it is also possible to design the hybridization chamber 3 in such a way that it can accommodate a DNS chip, so that the DNS chip can be replaced.

4 shows the basic sequence of the method according to the invention.

4A shows a field of a DNA array with a surface 11 to which oligonucleotides 12 which are not cleavable by nucleases and have the same nucleotide sequence in each field are bound.

4B shows how, after contacting this field with a sample solution, for example ^• an oligonucleotide 13 hybridizes from the sample solution to an oligonucleotide 12. This takes place with the formation of loops. In contrast, oligonucleotide 13 'binds in a regular manner to oligonucleotide 12 to form a double strand. The protruding part of the oligonucleotide 13 'in turn binds oligonucleotides 14 from the sample solution however form a disturbing background.

4C shows the result after digestion by S1 nuclease. The non-hybridized loops of the oligonucleotide 13 are now removed, as is the end of the nucleotide 13 'projecting beyond the oligonucleotide 12 and hybridized there oligonucleotides 14. Consequently, two short fragments 15, 16 and a correct double strand 17 remain.

4C shows the result after heating the oligonucleotide array to approximately 65 to 75 ° C. This destroys the S1 nuclease and the short fragments. 15, 16 detach from the oligonucleotide 12. This leaves only the correct double-stranded oligonucleotide 13 ', 17 and all further nucleotides 12 are for a further cycle to run through the steps ready according to Figures 4A to 4D.

After the completion of several such cycles, a sufficient number of oligonucleotides 12 are hybridized correctly double-stranded, so that the cycle is terminated and the double-stranded DNA is now stained with an intercalating dye. This enables an absolute quantification of the oligonucleotides from the sample that are hybridized in the field of the DNA array on the surface.

In a first example, it was demonstrated whether the VDR gene contains a point mutation that leads to a

Interface for the restriction endonuclease FokI leads. This point mutation is shown below, where a DNA segment with a total of 36 nucleotides is shown. C (mutation)

5'-CCCCTGCTCC TTCAGGGATG GAGGCAATGG CGGCCA -3 '(wild type)

T _M = 78.34 ° C.

A complementary oligonucleotide was prepared and immobilized to this wild-type DNA segment. The backbone of this oligonucleotide was modified as thiophosphonate as described above. As a result, it cannot be digested for the Sl nuclease M5761 from Promega.

If a wild-type genomic DNA fragmented from the DNA section binds to this oligonucleotide, a complete hybridization takes place with a melting point of T _M = 78.30 ° C. Since there is a complete hybridization and thus only a double strand, in the subsequent treatment step with the S1 nuclease mentioned only the supernatant at the 5 'or 3 "end, which is not hybridized with the oligonucleotide, is cut away, so that a 36 nucleotides of double-stranded DNA is formed.

However, if the mutation described is present, the hybridization takes place on 35 of the 36 nucleotides but not on the nucleotide that was exchanged. Here there is a single-stranded DNA, so that in the subsequent treatment step, the S1 nuclease cuts the hybridized DNA section at this point. The resulting DNA fragments are shown below

5'-C GCGGCCA-3 '

Furthermore, the S1 nuclease removes the protruding single-stranded DNA at both the 5 'and the 3' end. As a result, the immobilized oligonucleotide two DNA fragments hybridized with ≤ 18 nucleotides each. These two nucleotides have melting temperatures of

T _M = 64.4 ° C or T _M = 66.7 ° C.

If, for example, the temperature is then raised to approximately 70 ° C. in the subsequent step, the short sections detach from the immobilized oligonucleotide, while the wild-type DNA section remains hybridized. At the same time, the Sl nuclease is inactivated by this temperature increase step.

In the next step, again fragmented DNA can be added to the oligonucleotide arrangement. In this way, all oligonucleotides are saturated with wild-type DNA segments, if these are present, over the course of several cycles. In the subsequent detection using intercalating fluorescent dyes, it can thus be determined whether wild-type DNA or only DNA according to the mutation described above was present in the original sample.

In another example, the presence of a mutation in the VDR gene was investigated, in which an interface for the restriction endonuclease Bsml is formed. This is shown below

A (mutation) 5 '-GCCACAGACA GGCCTGCGCA TTCCCAATAC TCAG -3' (wild type)

T _M = 74.3 ° C

Here, too, an oligonucleotide is again produced and immobilized on the oligonucleotide arrangement, which is complementary to the nucleotide sequence of the wild-type DNA shown here. The further procedure proceeds as described in the example above, the fragments shown below arise when DNA of the mutation type binds to the oligonucleotide with a mismatch at the point mutation site and is then cut there by the S1 nuclease M5761.

5 '- TCAG -3'

The melting temperature of the hybridized widtype DNA section with 34 nucleotides is 74.3 ° C, while for the two fragments the melting temperatures are 64.4 ° C and 52.7 ° C, respectively.

If the above method is now carried out in the same way, only immobilized DNA fragments with 34 nucleotides of the wild type remain on the immobilized oligonucleotide, which is modified as above such that it cannot be cut by the S1 nuclease M5761. It can be used to demonstrate whether wild-type DNA was present in the original genomic DNA sample.

Of course, both examples can also be varied such that the immobilized nucleotide is complementary to. Sequence of the respective mutation, or different oligonucleotide sequences are immobilized on two fields of the oligonucleotide arrangement, one sequence being complementary to the wild type and the other sequence being complementary to the mutation. In this case, it is possible to make a precise distinction between homozygous wild type, homozygous mutation type or, if genomic DNA is detected, heterozygous wild type / mutation in both fields.

Claims

claims

1. oligonucleotide arrangement with a substrate which has at least one field, wherein in the at least one field oligonucleotides with a field-specific sequence are immobilized, characterized in that the immobilized oligonucleotides of the at least one field are modified in such a way that they counteract enzymatic, chemical, physical and / or chemical-physical degradation as a single strand and / or as a double strand are stable.

2. Oligonucleotide arrangement according to the preceding claim, characterized in that oligonucleotides are immobilized, which are not digestible by at least one predetermined or all nucleases that specifically cleave single-stranded nucleic acid.

3. Arrangement according to the preceding claim, characterized in that the oligonucleotides of the at least one field instead of a phosphoric acid ester bond in the sugar-phosphate backbone of the oligonucleotides contain thioesters.

4. Arrangement according to at least one of the preceding claims, characterized by at least two separate fields, wherein in the at least two separate fields oligonucleotides with field-to-field but different field-specific sequence are immobilized.

5. Arrangement according to at least one of the preceding claims, characterized in that the immobilized oligonucleotides are modified such that they hybridize with complementary nucleotide sequences in the same temperature range.

6. Arrangement according to the preceding claim, characterized in that the immobilized oligonucleotides are modified by suitable length or chemical modification, for example of the ribose component, that they hybridize with complementary nucleotide sequences in the same temperature range.

7. A method for the detection of a nucleotide sequence in a sample, characterized in that a) with at least sections of the nucleotide sequence to be detected complementary oligonucleotides are immobilized on a substrate, and the following steps b) and c) are carried out one or more times: b) this The substrate is brought into contact with the material of the sample; c) single-stranded DNA is broken down chemically, physically, chemico-physically, enzymatically or by another suitable method; and d) finally the presence of double-stranded nucleic acids or the nucleotide sequence to be detected is detected on the substrate.

8. The method according to the preceding claim, characterized in that between step b) and c) in a further step bl) the sub- strat is heated.

9. The method according to the preceding claim, characterized in that the substrate is heated to greater than 40 ° C.

10. The method according to the preceding claim, characterized in that the substrate is heated to 60 to 75 ° C.

11. The method according to any one of claims 7 to 10, characterized in that mobilized with at least portions of the detected nucleotide sequences complementary oligonucleotides on the substrate im- ^'ground that are modified such that they kleasen by a predetermined or all nucleic specifically single Split nucleic acid, cannot be digested.

12. The method according to any one of claims 7 to 11, characterized in that in step c) the surface of the substrate is subjected to a restriction digest by the predetermined or a nuclease that specifically cleaves single-stranded nucleic acid.

13. The method according to any one of claims 7 to 11, characterized in that in step c) the single-stranded DNA is chemically degraded.

14. The method according to the preceding claim, characterized in that in step c) the single-stranded DNA by means of hydroxylamine and osmium tetroxide followed by cleavage with piperidine or by hydrazine with piperidine or by dimethyl sulfoxide (DMS), formic acid and Piperidine is broken down.

15. The method according to any one of claims 7 to 14, characterized in that in each step b) further sample material is brought into contact with the substrate.

16. The method according to any one of claims 7 to 15, characterized in that non-inactivated nuclease is added before each step c).

17. The method according to any one of claims 7 to 16, characterized in that Sl-nuclease is used as nuclease.

18. The method according to any one of claims 7 to 17, characterized in that a sample containing fragmented, sheared, and / or previously partially digested and / or non-amplified nucleic acids is used as a sample.

19. The method according to any one of claims 7 to 17, characterized in that a sample containing amplified nucleic acid is used as a sample.

20. The method according to the preceding claim, characterized in that the amplified nucleic acids are labeled and in step d) labeled nucleic acid are detected on the substrate.

21. The method according to any one of claims 7 to 18, characterized in that for the detection of double-stranded nucleic acid in step d) the sub- strat dl) in one of the preceding steps or in step d) is brought into contact with a substance intercalating into double-stranded nucleic acids, d2) the substrate is washed and d3) the intercalating substance remaining on the substrate is detected.

22. The method according to the preceding claim, characterized in that a dye is used as the intercalating substance.

23. The method according to claim 22, characterized in that as an intercalating

A fluorescent dye is used.

24. The method according to claim 21, characterized in that a substance is used as the intercalating substance to which a marker, for example a radioactive component, a dye or a color reaction, a bioluminescence reaction and before or after the intercalation into the double-stranded nucleic acid / or an enzyme that catalyzes a chiluminescence reaction.

25. The method according to any one of claims 7 to 24, characterized in that for the detection of double-stranded nucleic acid in step d) d4) the substrate in one of the step preceding step d) or in step d) with a nucleic acid to be detected, but is not brought into contact with single-stranded mobilized oligonucleotide binding substance, d5) the substrate is washed d6) the substance remaining on the substrate and binding to the nucleic acid to be detected is detected.

26. The method according to the preceding claim, characterized in that a dye is used as the binding substance.

27. The method according to claim 25, characterized in that a fluorescent dye is used as the binding substance.

28. The method according to claim 25, characterized in that a substance is used as the binding substance to which a marker, for example a radioactive component, a dye or a color breakthrough, a bioluminescence reaction and before or after binding to the nucleic acid to be detected / or an enzyme catalyzing a chemiluminescent reaction can be bound.

29. The method according to any one of claims 25 to 28, characterized in that the substance which binds to the nucleic acid to be detected is a substance which binds, advantageously covalently, to the marginal sugar of the nucleic acid to be detected.

30. The method according to any one of the preceding claims, characterized in that for step d) the temperature of the substrate is below the hybridization temperature of the immobilized oligonucleotides with nucleotides complementary to them. is lowered.

31. The method according to any one of claims 7 to 30, characterized in that an oligonucleotide arrangement according to one of claims 1 to 6 is used.

32. Device for performing the method according to one of claims 7 to 31 with a holder for arranging an oligonucleotide arrangement according to one of claims 1 to 6 on a surface of the holder and a heating / cooling device for heating or cooling the oligonucleotide arrangement.

33. Device according to the preceding claim, characterized in that devices for supplying sample solutions, nuclease solutions, chemicals and / or washing solutions to the surface of the holder for the olionucleotide

Arrangement are present.

34. Device according to one of the two preceding claims, characterized by a spectral apparatus for

Detection of electromagnetic radiation arriving from above the surface of the holder, in particular X-rays, UV light, visible light, IR light or particle beams.

35. Device according to one of the three preceding claims, characterized by a device for radiation of electromagnetic radiation, in particular UV, visible and / or IR light on the surface of the bracket.