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WO2003072818A2 - Procede pour trier des acides nucleiques a un seul brin - Google Patents

Procede pour trier des acides nucleiques a un seul brin Download PDF

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Publication number
WO2003072818A2
WO2003072818A2 PCT/EP2003/002025 EP0302025W WO03072818A2 WO 2003072818 A2 WO2003072818 A2 WO 2003072818A2 EP 0302025 W EP0302025 W EP 0302025W WO 03072818 A2 WO03072818 A2 WO 03072818A2
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WO
WIPO (PCT)
Prior art keywords
stranded nucleic
nucleic acid
nucleic acids
sequences
seq
Prior art date
Application number
PCT/EP2003/002025
Other languages
German (de)
English (en)
Other versions
WO2003072818A3 (fr
Inventor
Dirk BÖKENKAMP
Tom Heike Dieck
Hans-Ulrich Hoppe
Original Assignee
Degussa Bioactives 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 Degussa Bioactives Gmbh filed Critical Degussa Bioactives Gmbh
Priority to AU2003214078A priority Critical patent/AU2003214078A1/en
Publication of WO2003072818A2 publication Critical patent/WO2003072818A2/fr
Publication of WO2003072818A3 publication Critical patent/WO2003072818A3/fr

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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

  • the present invention relates to a method for sorting single-stranded nucleic acids and the use thereof.
  • genes with a defined nucleic acid sequence are active in a typical human cell, which always characterize the current state of the cell.
  • the state of a cell indicates an increased cell division activity (reproductive cell;
  • Cancer cell or generally altered metabolic activities as they occur in the event of illness or usually with aging.
  • the genetic state of the cell also reacts to exogenous causes, such as changes in eating habits or intoxications.
  • the activity of a specific gene can be, for example, via its mRNA
  • Translation product can be determined.
  • the mRNA or protein pattern of a cell therefore reflects its current state very precisely.
  • the characterization of the nucleic acid profile of cells has recently come to the fore, particularly in connection with the screening of pharmacologically active compounds. Numerous methods for characterizing the genetic state of a cell and its protein profile have already been developed.
  • arrays are defined by certain arrangements of immobilized recognition species, which in the meantime play an important role in diagnostics, among others, in the simultaneous one Play determination of so-called analytes. Examples are typical nucleic acid arrays as described in Southern et al., Genomics (1992) 13, 1 008, or in patents US 5,631, 1 34 or US 5,795,71 4.
  • No. 5,795,714 describes a method for replicating an array of nucleic acid samples that can be used on an industrial scale in the production of diagnostics for screening biological samples with special target sequences.
  • a first set of nucleic acids is first synthesized, which has a constant sequence of a certain length at the 3 'end and a random sequence of a certain length at the 5' end; then a second set of nucleic acids is synthesized with sequences complementary to the constant sequence of the first set of nucleic acids; finally, the first set of nucleic acids is hybridized with the second set of nucleic acids and thus the desired array of probes is obtained which, in addition to a double-stranded region, also have a single-stranded region which in turn has the random sequence.
  • the object of the present invention is therefore to provide a method for sorting single-stranded nucleic acids in which the disadvantages of the prior art are at least partially eliminated. With this method it is possible in particular With the help of an inexpensive and fast method and in a technically relatively simple way, the entire pattern of single-stranded nucleic acids of a cell can be reproduced.
  • the capture substances are oligonucleotides with 5'-terminal invariable anchor sequences which occur in complementary form at least once in each single-stranded nucleic acid, and with 3 ' -terminal variable discrimination sequences, consisting of several different sequences, preferably each possible sequence of ⁇ 10 nucleotides, which enable the formation of certain types of single-stranded nucleic acids.
  • this method when using this method, it has been found that the entire and thus complete pattern of all single-stranded nucleic acids in a cell can be reproduced reproducibly, and a diagnostic determination of the status of the cell under investigation is possible in this way.
  • this method can not only be used to screen pharmaceutical and / or bioactive compounds, but also in the medical field the finest changes in the nucleic acid pool of cells, in particular of cells with a high division rate, can be shown become. The variety of these possibilities could not have been foreseen.
  • An essential aim of the method according to the present invention is the formation of certain types of single-stranded nucleic acids.
  • the term “variety” is intended to encompass those single-stranded nucleic acids which occupy a defined distribution site on the respective matrix system (for example a chip).
  • This type of nucleic acid is characterized by the fact that it is a shows selective hybridization with a specific capture substance under the chosen test conditions.
  • the various types of single-stranded nucleic acids present in the sample, which are separated by the method according to the invention, preferably form a "pattern" in their entirety which is characteristic of the condition of a cell.
  • the method according to the invention should therefore not be used to determine specific nucleic acid sequences, e.g. Sequences of a cell transcriptome are identified and displayed, but rather different patterns are to be formed which reflect a certain (current) state of the transcriptome of a cell.
  • the focus of the method according to the invention is therefore an assignment of nucleic acid patterns and not the mapping of individual genes, which is why matrix miniaturization is also possible.
  • a complete distribution of the single-stranded nucleic acids present in the sample can be achieved on an array.
  • capture substances in the form of oligonucleotides or nucleic acid analogs are used, which cover all potentially possible sequences.
  • the different nucleic acid pools are then hybridized on different matrix platforms (chips) and the resulting patterns are compared with one another.
  • the resulting pattern of the nucleic acid hybridization event can be displayed via suitable detection systems (for example fluorescence).
  • the present method uses a transcriptome, which is in particular RNA and / or DNA.
  • RNA and / or DNA are in particular RNA and / or DNA.
  • all types come here single-stranded conveniences, such as RNA in general or mRNA, cRNA, pRNA, DNA, such as cDNA, or nucleic acid analogues, such as PNA, LNA or combinations thereof.
  • capture substances capture probes
  • these capture molecules must have two sections in order to be suitable as such:
  • Nucleic acids are "captured", and (ii) a variable and distribution-dependent on the matrix
  • Section that has a discriminatory effect on single-stranded nucleic acids is a section that has a discriminatory effect on single-stranded nucleic acids.
  • variable sequence means a single-stranded oligonucleotide section, but also a single-stranded sequence of oligonucleotide analogs, each with a defined nucleotide sequence.
  • variable sequence is understood to mean nucleic acid sequences, the sequence of which in complementary form has several different, e.g. > 10, preferably> 20, particularly preferably> 50 sequences and most preferably covers all potentially possible sequences of the single-stranded nucleic acids to be sorted.
  • nucleic acid sequences with a length of 10 to 50 nucleotides and in particular of 15 to 25 nucleotides are used as anchor sequences of the capture substances.
  • An oligo-dT sequence for example for immobilizing polyA-RNA molecules or an oligo-dA sequence, is particularly preferred as anchor sequences Immobilization of polyT-containing cDNA molecules.
  • Other anchor sequences can be consensus sequences or conserved areas of certain gene classes.
  • the capture substances are preferably regularly distributed in nucleotide analogs, e.g. So-called “Locked Nucleic Acids” (LNA), whereby otherwise normal nucleotide sequences at mostly certain locations (e.g. every third position) contain a special nucleotide, which gives the capture substance high affinity properties. This can increase the binding capacity to the single-stranded nucleic acids in the sample to be examined.
  • LNA Locked Nucleic Acids
  • the 3'-terminal, variable sequences of the catcher have the actual discrimination function for cultivar formation.
  • the invention provides that combinations of the nucleic bases adenine, cytosine, guanine, thymine, uracil, inosine and their analogues are used as preferred 3'-discrimination sequences, the length of which 3 Discrimination sequences are advantageously from 3 to 8 nucleotides.
  • the length of the discriminating sequence can also be longer, which of course also results in a higher resolution of the distribution pattern.
  • matrix is understood to mean materials in solid or gel-like form.
  • plastics, ceramics, glasses, metals, such as e.g. Precious metals, crystalline materials and / or membranes, each with semiconductor properties are suitable. These can also be in the form of thin layers.
  • a matrix in chip form is to be regarded as particularly preferred.
  • the single-stranded nucleic acids are selectively bound with the aid of the capture substance, which itself is immobilized, that is to say bound, on the matrix surface.
  • the capture substances are preferred according to the invention via a bioaffine interaction between two partners of a binding pair, e.g. Biotin / streptavidin or avidin or hapten / antibodies etc., bound to the matrix surface.
  • the capture substances in biotinylated form are particularly preferably bound to the matrix surface by means of streptavidin, which preferably represents a layer of (bio) molecular filaments, such as cellulose and agarose, framework proteins, hydrogels or polyacrylamide.
  • the capture substances are immobilized in a site-specific manner, for example concentrated and immobilized by means of tension at the positions of the array intended for them. This definition happens in a known manner by binding the biotin residues, which are at the 5 'end, with streptavidin, which is fixed in the matrix surface.
  • Carrier systems in the form of semiconductor chips are known to be very well suited to electronically controllable specific binding reactions of nucleic acids at fixed, addressable locations in the form of an array.
  • the present invention also provides that the capture substances are bound to the matrix surface covalently, quasi-covalently, supramolecularly, physically, in an electrical field or by means of a molecular sieve.
  • nucleic acid pool (analyte) of a sample to be sorted on a nucleic acid array for example on a semiconductor chip
  • the nucleic acid array for example on a semiconductor chip
  • the bound nucleic acids by simply reversing the polarity of the electrical field be removed.
  • Electronic chip variants are therefore particularly suitable.
  • the final readout of the fixed single-stranded nucleic acids provided for the present method is carried out by displaying the now sorted nucleic acid fractions as reproducible patterns.
  • the present invention also sees the actual method for sorting single-stranded nucleic acids Use of this method before, the analysis of single-stranded nucleic acid patterns, in particular in mucous membrane, hair root, blood, nerve or reproductive cells, to be regarded as preferred.
  • the present invention also provides the use of the claimed method for the detection of the activity of pharmaceutically and / or nutritionally active compounds, such as food additives or supplements, in particular of minerals, trace elements, organic acids, such as amino acids, carboxylic acids and fatty acids, and their derivatives, salts and mixtures.
  • pharmaceutically and / or nutritionally active compounds such as food additives or supplements, in particular of minerals, trace elements, organic acids, such as amino acids, carboxylic acids and fatty acids, and their derivatives, salts and mixtures.
  • the animals were 24 male Sprague-Dawley rats with an average weight of. 1 21 g ( ⁇ 9.5), which were divided into two groups with 1 2 animals each. All animals received a semi-synthetic diet (AIN 93G) four times a day by gavage.
  • the diet of the control group (“untreated") contained 25 mg Zn / kg diet, that of the deficient group (“treated”) 1.3 mg Zn / kg diet.
  • the animals were decapitated after light anesthesia and the blood was collected and preserved in heparinized tubes for biochemical analysis.
  • the tissue samples taken were snap frozen and stored at -80 ° C.
  • the total RNA from the liver was isolated using a modified guanidinothiocyanate / phenol / chloroform extraction.
  • NanoChip TM cartridge from Nanogen, San Diego (USA) was used as the micro-array, the electronic semiconductor chip of which has 100 (1 x 10) occupied spaces and 100 individually controllable electrodes (maximum charge density: approx. 10 9 fragments per Area; chip size: 0.7 cm 2 ; array size: 2 mm 2 ).
  • NanoChip TM Molecular Biology Workstation from Nanogen, San Diego (USA), which has a highly sensitive laser-based fluorescence scanner, was used to read out the nucleic acid patterns. It has two lasers, with excitation wavelengths of 635 nm (Laser I) and 532 nm (Laser II).
  • Detection was carried out using a photomultiplier method (radiation wavelength I: 660 to 720 nm (Cy 5); emission wavelength II: 550 to 600 nm (Cy 3)).
  • the 64 capture substances in the form of oligonucleotides consisting of 1 8mer dT and 3 discriminating bases (see Table 1), which had a so-called Locked Nucleic Acid (LNA) at every third T base site instead of a normal deoxyribonucleotide Concentrated and immobilized for 1 min at the intended positions of the array using voltage (2.0 volts) (for distribution see Table 2).
  • the biotinylated capture substances were immobilized by binding via streptavidin in the agarose layer of 2 chips as the carrier matrix. After immobilization of the capture probes, the electronic hybridization took place.
  • the unlabelled polyA-RNA samples of the treated and untreated animals were hybridized on the two chips for 2 minutes and under 2.1 volts.
  • a series of discrimination was carried out in the device using a counter voltage of -0.5 to -1.08 volt in order to remove non-specifically bound molecules.
  • a so-called Enzymatic Reporting was carried out with the incorporation of fluorescence-labeled nucleotides (Cy 5-CTP) for labeling.
  • the pattern was then fluorescence-optically displayed by laser excitation in the reader (see Figure 3).
  • RNA single-stranded nucleic acid
  • the distribution of the capture substances is shown on a chip with 1 0 x 10 filling positions; columns 1 and 2 contain sequence-specific control gene oligonucleotides; empty fields mean vacant chip spaces.

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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé permettant de trier des acides nucléiques à un seul brin par isolement et hybridation de réservoirs d'acide nucléique appropriés et par sélection subséquente. Les acides nucléiques à un seul brin sont liés de manière sélective à l'aide de substances de capture (capture) immobilisées sur une surface de matrice fixe, qui présentent des zones variables et invariables. Ce procédé se caractérise en ce que les substances de capture sont des oligonucléotides à séquences d'ancrage 5'-terminales invariables, se présentant sous forme complémentaire au moins une fois dans chaque acide nucléique à un seul brin, ainsi qu'à séquences de discrimination 3'-terminales variables, consistant en toute suite possible de ≤10 nucléotides, qui permettent la formation de catégories déterminées d'acides nucléiques à un seul brin. Outre ledit procédé, l'invention concerne son utilisation pour analyser des modèles d'acide nucléique à un seul brin, notamment dans des cellules de la muqueuse, de la racine des cheveux, dans des cellules sanguines, des cellules nerveuses ou dans des cellules de reproduction, ainsi que l'utilisation pour détecter l'effet de composés à action pharmaceutique et/ou relevant de la nutrition physiologique. Ce nouveau procédé permet de représenter de manière rapide et économique, des différences reproductibles dans un réservoir d'acide nucléique à un seul brin de cellules.
PCT/EP2003/002025 2002-02-28 2003-02-27 Procede pour trier des acides nucleiques a un seul brin WO2003072818A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003214078A AU2003214078A1 (en) 2002-02-28 2003-02-27 Method for sorting single-stranded nucleic acids

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002108794 DE10208794A1 (de) 2002-02-28 2002-02-28 Verfahren zum Sortieren einsträngiger Nucleinsäuren
DE10208794.6 2002-02-28

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WO2003072818A2 true WO2003072818A2 (fr) 2003-09-04
WO2003072818A3 WO2003072818A3 (fr) 2004-03-11

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DE (1) DE10208794A1 (fr)
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Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
DE69333650T2 (de) * 1992-02-19 2006-01-12 The Public Health Research Institute Of The City Of New York, Inc. Neue anordnungn von oligonukleotiden und ihr nutzen zum sortieren, isolieren, sequenzieren und manipulieren von nukleinsäuren
WO2000026412A1 (fr) * 1998-11-02 2000-05-11 Kenneth Loren Beattie Analyse d'acides nucleiques par hybridation en tandem ciblee sur des sequences
KR20020007332A (ko) * 1999-03-18 2002-01-26 추후제출 일 단계 샘플 제조 및 복합적인 생물학적 샘플에서 핵산의검출
ATE408141T1 (de) * 1999-10-20 2008-09-15 Shigeori Takenaka Gen-detektionschip, sensor und nachweisverfahren
DE60114525T2 (de) * 2000-07-31 2006-07-20 Agilent Technologies Inc., A Delaware Corp., Palo Alto Array-basierende Methoden zur Synthese von Nukleinsäuregemischen
EP1186673A3 (fr) * 2000-09-11 2003-03-26 Agilent Technologies, Inc. (a Delaware corporation) Calibration des dates d'origine des réseaux moléculaires

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WO2003072818A3 (fr) 2004-03-11
DE10208794A1 (de) 2003-09-04
AU2003214078A1 (en) 2003-09-09

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