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WO2003048385A2 - Structure oligonucleotidique, detection de nucleotides et dispositif associe - Google Patents

Structure oligonucleotidique, detection de nucleotides et dispositif associe Download PDF

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Publication number
WO2003048385A2
WO2003048385A2 PCT/EP2002/013861 EP0213861W WO03048385A2 WO 2003048385 A2 WO2003048385 A2 WO 2003048385A2 EP 0213861 W EP0213861 W EP 0213861W WO 03048385 A2 WO03048385 A2 WO 03048385A2
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WO
WIPO (PCT)
Prior art keywords
substrate
oligonucleotides
detected
nucleic acid
substance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2002/013861
Other languages
German (de)
English (en)
Other versions
WO2003048385A3 (fr
Inventor
Stefanie WASCHÜTZA
Alf-Andreas Krehan
Oliver BÖCHER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alere Diagnostics GmbH
Original Assignee
Adnagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Adnagen GmbH filed Critical Adnagen GmbH
Priority to AU2002349054A priority Critical patent/AU2002349054A1/en
Priority to EP02781342A priority patent/EP1456406A2/fr
Publication of WO2003048385A2 publication Critical patent/WO2003048385A2/fr
Publication of WO2003048385A3 publication Critical patent/WO2003048385A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ l with an area of 4 qcm, this is reduced to approx. 1 ng).
  • the hybridization solution is replaced by a solution for restriction digestion with S1 nuclease, the temperature being reduced to 37 ° C.
  • S1 nuclease M5761 from Promega was used here as an example of the Sl nuclease.
  • 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.
  • remaining short DNA sections which, however, do not hybridize with the entire oligonucleotide, are detached without detaching completely hybridized DNA sections to be detected.
  • 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.
  • 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.
  • 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:
  • 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.
  • a selective step of breaking down, for example restriction cleavage on the array, is carried out;
  • Stabilized immobilized oligonucleotides are advantageously used to enable repeated hybridizations
  • the intercalation dye can only be added at the end of the process, thereby avoiding light-protected handling during the previous hybridizations.
  • 4 figures show a device according to the invention and the basic process sequence.
  • Figure 1 shows a device according to the invention in cross section.
  • FIG. 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
  • Fig. 4 shows the basic process flow of the method according to the invention.
  • 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.
  • 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.
  • a hybridization chamber 3 which is designed as a DNS chip, is also introduced into a carrier 2.
  • 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.
  • the hybridization chamber 3 in such a way that it can accommodate a DNS chip, so that the DNS chip can be replaced.
  • 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.
  • FIG. 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.
  • 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 oli
  • 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.
  • 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.
  • VDR gene contains a point mutation that leads to a
  • 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.
  • the hybridization takes place on 35 of the 36 nucleotides but not on the nucleotide that was exchanged.
  • the S1 nuclease cuts the hybridized DNA section at this point.
  • the resulting DNA fragments are shown below
  • the S1 nuclease removes the protruding single-stranded DNA at both the 5 'and the 3' end.
  • the immobilized oligonucleotide two DNA fragments hybridized with ⁇ 18 nucleotides each. These two nucleotides have melting temperatures of
  • 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.
  • 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.
  • 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.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
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  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Structure oligonucléotidique qui comporte un substrat possédant au moins un champ dans lequel sont immobilisés des oligonucléotides à séquence spécifique de champ. Les oligonucléotides immobilisés d'un (des) champ(s) sont modifiés de manière telle qu'ils sont stables en tant que brin ou unique et / ou en tant que double brin par rapport à une dégradation enzymatique, chimique, physique et / ou physico-chimique.
PCT/EP2002/013861 2001-12-06 2002-12-06 Structure oligonucleotidique, detection de nucleotides et dispositif associe Ceased WO2003048385A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002349054A AU2002349054A1 (en) 2001-12-06 2002-12-06 Oligonucleotide array, nucleotide detection method and device therefor
EP02781342A EP1456406A2 (fr) 2001-12-06 2002-12-06 Structure oligonucleotidique, detection de nucleotides et dispositif associe

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10159904A DE10159904A1 (de) 2001-12-06 2001-12-06 Oligonukleotidanordnung, Verfahren zum Nukleotidnachweis sowie Vorrichtung hierfür
DE10159904.8 2001-12-06

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Publication Number Publication Date
WO2003048385A2 true WO2003048385A2 (fr) 2003-06-12
WO2003048385A3 WO2003048385A3 (fr) 2004-04-08

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PCT/EP2002/013861 Ceased WO2003048385A2 (fr) 2001-12-06 2002-12-06 Structure oligonucleotidique, detection de nucleotides et dispositif associe

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EP (1) EP1456406A2 (fr)
AU (1) AU2002349054A1 (fr)
DE (1) DE10159904A1 (fr)
WO (1) WO2003048385A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006114995A1 (fr) * 2005-04-20 2006-11-02 Sony Corporation Procede de detection d’hybridisation par l’utilisation d’intercaleur

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Publication number Priority date Publication date Assignee Title
US5378825A (en) * 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
DE69126530T2 (de) * 1990-07-27 1998-02-05 Isis Pharmaceutical, Inc., Carlsbad, Calif. Nuklease resistente, pyrimidin modifizierte oligonukleotide, die die gen-expression detektieren und modulieren
EP0558740B1 (fr) * 1991-09-19 1998-08-05 VYSIS, Inc. Composition de sondage destinee a l'identification de genomes, et procedes associes
EP1044987B1 (fr) * 1991-12-24 2006-02-15 Isis Pharmaceuticals, Inc. Oligonucléotides modifiés en 2' à ouverture
DE4338704A1 (de) * 1993-11-12 1995-05-18 Hoechst Ag Stabilisierte Oligonucleotide und deren Verwendung
DE19633436A1 (de) * 1996-08-20 1998-02-26 Boehringer Mannheim Gmbh Verfahren zum Nachweis von Nukleinsäuren unter Ermittlung der Masse
EP1060266A4 (fr) * 1998-02-23 2004-09-22 Dana Farber Cancer Inst Inc Procede permettant d'identifier les sites reactifs des glycosylases de reparation des mesappariements, compose et utilisations de ce dernier
EP1259524A2 (fr) * 1999-03-09 2002-11-27 Amersham Biosciences UK Limited Nucleotides et polynucleotides immobilisees sur un support solide au moyen de liaison disulfure
EP1077264B1 (fr) * 1999-06-07 2005-04-20 Fuji Photo Film Co., Ltd. Puce d'ADN, puce d'APN, et des procédés de préparation
US6528319B1 (en) * 1999-09-02 2003-03-04 Amersham Biosciences Corp Method for anchoring oligonucleotides to a substrate
US6608036B1 (en) * 1999-09-10 2003-08-19 Geron Corporation Oligonucleotide N3′→P5′ thiophosphoramidates: their synthesis and administration to treat neoplasms
DE10010282B4 (de) * 2000-02-25 2006-11-16 Epigenomics Ag Verfahren zur Detektion von Cytosin-Methylierung in DNA Proben
DE10019136A1 (de) * 2000-04-18 2001-10-31 Aventis Pharma Gmbh Polyamidnukleinsäure-Derivate, Mittel und Verfahren zu ihrer Herstellung
DE10021204A1 (de) * 2000-04-25 2001-11-08 Epigenomics Ag Verfahren zur hochparallelen Analyse von Polymorphismen

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006114995A1 (fr) * 2005-04-20 2006-11-02 Sony Corporation Procede de detection d’hybridisation par l’utilisation d’intercaleur

Also Published As

Publication number Publication date
DE10159904A1 (de) 2003-07-03
EP1456406A2 (fr) 2004-09-15
AU2002349054A1 (en) 2003-06-17
WO2003048385A3 (fr) 2004-04-08

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