EP2217364A2 - Procédé d'extraction flexible permettant la production de bibliothèques de molécules spécifiques d'une séquence - Google Patents

Procédé d'extraction flexible permettant la production de bibliothèques de molécules spécifiques d'une séquence

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Publication number
EP2217364A2
EP2217364A2 EP08851271A EP08851271A EP2217364A2 EP 2217364 A2 EP2217364 A2 EP 2217364A2 EP 08851271 A EP08851271 A EP 08851271A EP 08851271 A EP08851271 A EP 08851271A EP 2217364 A2 EP2217364 A2 EP 2217364A2
Authority
EP
European Patent Office
Prior art keywords
molecules
capture
target molecules
sample
support
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.)
Withdrawn
Application number
EP08851271A
Other languages
German (de)
English (en)
Inventor
Markus Beier
Stephan Bau
Daniel Summerer
Mark Matzas
Peer STÄHLER
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.)
Febit Holding GmbH
Original Assignee
Febit Holding 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 Febit Holding GmbH filed Critical Febit Holding GmbH
Publication of EP2217364A2 publication Critical patent/EP2217364A2/fr
Withdrawn legal-status Critical Current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

  • the invention relates to an extraction method for isolating target molecules from a sample using a library of molecules. o
  • the selective extraction of molecules is a central and important process in many areas of biochemistry, biology and medicine. These areas include the extraction and purification of nucleic acids, proteins, sugars and other biochemical functional molecules. 5
  • nucleic acids usually DNA and RNA.
  • Essential steps of recombination techniques are to isolate and purify certain nucleic acids, e.g. of plasmid DNA or genomic DNA.
  • mRNA messenger RNA
  • Biochemical, biological and medical analysis methods can be enormously increased in their efficiency and significance by miniaturization and parallelization.
  • miniaturization relates, for example, to the analysis of genetic material by means of hybridization experiments on DNA microarrays.
  • DNA probes as receptors
  • methods have also been miniaturized and parallelized, which serve to screen for molecules with special properties, eg, ribozymes.
  • receptors on microarrays are proteins and those molecules that are not found in nature, such as peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • Many such assay formats are grouped under the Biochips heading. All of these assay formats and biochips are potentially useful for isolation and purification of sample material and sample preparation.
  • Microreaction techniques can be coupled with such microarrays to provide fast and efficient systems both in sample preparation and in the fabrication of the actual array. This also includes the use of microfluidic methods.
  • mRNA molecules can be isolated relatively specifically by growing on a solid phase, e.g. Latex beads, magnetic beads, controlled pore glass beads or the matrix of a column immobilized poly-thymidine strands (poly-T strands). These poly-T strands, upon addition of total RNA, hybridize with the poly-adenine (poly-A) tail of mRNA molecules and allow separation of the unbound RNA molecules. The mRNA molecules are then isolated by appropriately changing the buffer so that hybridization in favor of single strands is abolished. The liberated mRNA molecules can then be eluted.
  • a solid phase e.g. Latex beads, magnetic beads, controlled pore glass beads or the matrix of a column immobilized poly-thymidine strands (poly-T strands).
  • poly-T strands upon addition of total RNA, hybridize with the poly-adenine (poly-A) tail of mRNA molecules and allow separation of the unbound RNA molecules.
  • the information content of such an isolation matrix is comparatively small, since only two categories of target molecules can be distinguished, namely with or without poly-A tail.
  • immunoglobulins are often isolated by an isolation matrix in an immobilized Protein A (from Staphylococcus aureus) column (Brown et al., Biochem. Soc. Transactions (England) 26 (1998), 249).
  • proteins from Staphylococcus aureus
  • antibodies to one or a few target molecules are bound to the isolation matrix.
  • U.S. Patent 6,013,440 describes a method of making an affinity matrix wherein a set of different nucleic acid probes are immobilized on a solid support to thereby enrich target nucleic acids of a still unknown sequence from a sample.
  • the proposed method uses sheet carriers as the isolation matrix, allowing only unfavorable elution. Even with this method, the known from the prior art disadvantages can not be eliminated.
  • WO 03/031965 describes a method and an apparatus in which a microfluidic carrier is used as the isolation matrix, which allows a selective isolation of certain biochemical functional molecules (target molecules), in particular a sequence-specific parallel isolation of multiple species of target molecules from a mixture.
  • a microfluidic carrier is used as the isolation matrix, which allows a selective isolation of certain biochemical functional molecules (target molecules), in particular a sequence-specific parallel isolation of multiple species of target molecules from a mixture.
  • the present invention relates to a method of isolating target molecules from a sample using a library of molecules comprising free capture receptors that are specific - A -
  • the method preferably comprises parallel isolation of multiple target molecules using a library of several different capture receptors.
  • the synthesis of the molecule library takes place on a solid support, in particular on a microfluidic support.
  • the catcher receptors forming the molecule library are detached from the carrier, in particular by chemical or / and physical methods, or copied off or copied off, in particular by enzymatic methods.
  • the capture receptors are tagged with functional groups and then contacted with the sample so that binding of the target molecules to targeting receptor specific receptors can occur and complexes of target molecules and capture receptors are formed.
  • the complexes formed e.g. consist of a capture receptor and a target molecule are then separated from remaining sample components, preferably by immobilization to a solid phase, e.g. about the functional group. Subsequently, the target molecules bound to the solid phase can be eluted.
  • the functionality of a target molecule is defined by its ability to selectively bind to a targeting molecule-specific capture receptor, preferably through bioaffinity interactions such as hybridization, receptor-ligand binding, antigen-antibody binding, saccharide-lectin binding, etc.
  • the method according to the invention comprises the provision of a carrier with a molecule library, ie an array of selectively binding receptors or capture probes which are suitable for isolating the target molecules.
  • a carrier ie an array of selectively binding receptors or capture probes which are suitable for isolating the target molecules.
  • an in situ synthesis of the receptors takes place on or in the carrier, preferably a microfluidic reaction carrier.
  • This procedure allows for the use of appropriate methods for in situ synthesis a very high information content of the molecule library.
  • nucleic acids as Target molecules can make a suitable system for the in situ synthesis of the corresponding capture probes thousands of defined sequences of a molecule library.
  • the invention provides a way to selectively isolate hundreds to thousands or even millions of individual DNA or RNA molecules from a mixture.
  • a variation of the method described above is to use the sample containing several different target molecules, e.g. Nucleic acids, to contact with a carrier having reversibly bound different freely selectable capture molecules. After removal of unbound or insufficiently bound components of the sample, the complexes of target molecules and capture receptors are detached from the carrier. Subsequently, a separation of the target molecules from the capture receptors, e.g. via functional groups of capture receptors that allow biophysical separation.
  • target molecules e.g. Nucleic acids
  • the method of the invention may e.g. They are used in academic research, basic research, industrial research, quality control, pharmaceutical research, biotechnology, clinical research, clinical diagnostics, screening, patient-specific diagnostics, and clinical trials , in forensics, for genetic testing, such as parenting, in animal and plant breeding or in environmental monitoring.
  • An object of the invention is thus a method for isolating target molecules from a sample, comprising the steps:
  • step (c) labeling the capture molecules, wherein marking occurs before or after Detachment of the capture molecules according to step (b) can take place
  • Another object of the invention is a method for the isolation of target molecules from a sample, comprising the steps: (a) providing a support with an array of several different optional catcher molecule matrices immobilized at respectively different positions on or in the support (b) copying the capture molecule templates to obtain capture molecules in free form,
  • step (c) marking the catcher molecules, wherein the marking can take place during or after the copying according to step (b),
  • Target molecules can specifically bind to the labeled capture molecules
  • Yet another subject of the invention is a method for isolating target molecules from a sample comprising the steps:
  • the target molecules isolated by the method of the invention are preferably selected from biological polymers such as nucleic acids, e.g. double-stranded or single-stranded DNA molecules, e.g. genomic DNA molecules or cDNA molecules, or RNA molecules, polypeptides such as proteins, glycoproteins, lipoproteins, nucleoproteins, etc., peptides and saccharides.
  • biological polymers such as nucleic acids, e.g. double-stranded or single-stranded DNA molecules, e.g. genomic DNA molecules or cDNA molecules, or RNA molecules, polypeptides such as proteins, glycoproteins, lipoproteins, nucleoproteins, etc., peptides and saccharides.
  • the target molecules are nucleic acids and the capture molecules are selected from hybridization probes complementary thereto.
  • the hybridization probes can likewise be nucleic acids, in particular DNA molecules, but also nucleic acid analogs, such as peptide nucleic acids (PNA) 1 Locked nucleic acids (LNA), etc.
  • PNA peptide nucleic acids
  • LNA Locked nucleic acids
  • the hybridization probes preferably have a length corresponding to 10-100 nucleotides and do not necessarily consist of building blocks with bases, ie they may also contain, for example, abasic building blocks, linkers, spacers, etc.
  • the hybridization probes or their complements may be attached to the support at the 3'-end, 5'-end or therebetween or at several positions.
  • capture molecules are peptides.
  • low molecular weight substance libraries can be used as receptors.
  • the capture molecules or capture molecule matrices immobilized on the support are generated on the support by in situ synthesis, for example by stepwise synthesis from synthesis building blocks, and are thus freely selectable.
  • the building blocks for this in situ synthesis may be monomers of the relevant substance class, ie, in the case of nucleic acids, for example nucleotide building blocks. However, it may also be more complex components, such as oligonucleotides or oligopeptides of several, for example, 2, 3, or 4, monomer units.
  • the sample used for the method according to the invention is preferably a complex sample, i. the target molecules must be selectively isolated from a variety of similar molecular species.
  • the sample may be a biological sample, e.g. a sample from a biological organism, e.g. from a body fluid, a sample from a cell or microorganism culture, etc.
  • the sample may also be made from synthetic sources, e.g. from a synthesizer, or be a mixture of biological and synthetic material.
  • synthetic sources e.g. from a synthesizer
  • the use of prefabricated molecular libraries in which specific target molecules may be present is also possible.
  • the sample may optionally be processed prior to contacting the receptors, e.g. by enzymatic reaction such as amplification, restriction cleavage, labeling, transcription, translation, fractionation, pre-purification etc.
  • enzymatic reaction such as amplification, restriction cleavage, labeling, transcription, translation, fractionation, pre-purification etc.
  • the target molecules to be isolated can thus be present in labeled or unlabeled form.
  • the array containing the capture molecules or capture molecule matrices in immobilized form is preferably provided on a structured support, more preferably on a support with channels, for example with closed channels.
  • the channels are, for example, microchannels with a cross section of 10-10,000 microns.
  • suitable carriers with channels are described in WO 00/13017 and WO 00/13018.
  • a carrier is used which is at least partially in the range the positions with the immobilized capture molecules or capture molecule matrices is optically transparent and / or electrically conductive.
  • other types of supports for example planar supports such as microscope slides, can be used, which allow the preparation of defined molecule libraries of capture molecules.
  • the support used for the method of the invention contains an array of several different species of immobilized capture molecules or capture molecule templates, e.g. with at least 10, preferably at least 100, particularly preferably at least 1000 different species of catcher molecules or catcher molecule matrices.
  • the individual immobilized species differ in that they have a different structure, e.g. Nucleic acid sequence, and optionally - in the case of catcher molecules - bind different target molecules and - in the case of catcher molecule matrices - result in the write off different catcher molecules.
  • the capture molecules or capture molecule matrices are preferably built up in situ stepwise by local or / and time-specific coupling of synthesis building blocks at the respectively predetermined positions on or in the support.
  • a single capture molecule species or capture molecule template species or a mixture of several different capture molecule species or capture molecule template species may be immobilized or synthesized.
  • the capture molecules can also be synthesized externally and then immobilized on the support, e.g. by spotting.
  • the carrier is integrated in a device comprising a programmable light source matrix, a Detector matrix, a preferably arranged between the light source and detector matrix carrier and means for supplying fluids into the carrier and for discharging fluids from the carrier.
  • the programmable light source or exposure matrix may be a reflection matrix, a light valve matrix, eg, an LCD matrix or a self-emitting exposure matrix.
  • Such light matrices are disclosed in WO00 / 13017 and WO00 / 13018.
  • the detector matrix can be integrated in the carrier body.
  • the in situ buildup of capture molecules on the support can include fluid chemical steps, photochemical steps, electrochemical steps, or combinations of two or more of these steps.
  • An example of an electrochemical synthesis of catcher molecules on a carrier is described in DE 101 20 633.1.
  • An example of a hybrid process comprising the combination of fluid chemical steps and photochemical steps is described in DE 101 22 357.9.
  • the inventive method is also suitable for the production of multiple libraries of capture molecules on a single carrier.
  • catcher molecules after the replacement of catcher molecules or after copying catcher molecule matrices another synthesis Abansviz. Connect write-down cycle.
  • very different molecular species such as mRNA molecules or DNA sequences, can be isolated with a carrier.
  • a switch to new target molecules with a single carrier is possible.
  • the method is easy to automate and can be combined with further subsequent processing steps, such as an amplification reaction, e.g. using a PCR thermocycler, detection or in vitro translation.
  • the capture molecules which can specifically bind to the target molecules are provided directly on the support.
  • the capture molecules which can specifically bind to the target molecules are provided directly on the support.
  • the capture molecules are coupled via a cleavable bond to the carrier, so that a detachment of the capture molecules from the carrier can follow, for example, by photochemical and / or fluid chemical steps.
  • photolabile and / or chemically labile e.g. provided by acids, bases and / or reduction cleavable bonds between the capture molecule and the carrier.
  • the capture molecules are marked. This is preferably done by introducing one or more functional groups. These functional groups can be introduced, for example, during synthesis in the form of functionalized synthesis building blocks and / or after synthesis (on the support or after release) by reaction of the capture molecule with suitable functional groups.
  • a carrier with immobilized capture molecule matrices is provided.
  • the capture molecules are prepared in free form, for example by enzymatic elongation of primer molecules which bind to partial sequences of the templates, e.g. hybridize, or by other methods, such as described in WO 2005/051970, the disclosure of which is hereby made the subject of the present application.
  • the capture molecule matrices are preferably nucleic acids and the capture molecules thereof complementary nucleic acid molecules.
  • the introduction of tag groups into the capture molecules may occur during preparation, e.g. during the enzymatic synthesis, for example by using primer molecules derivatized with functional groups and / or synthesis building blocks, or after the capture molecules have been prepared by derivatization with suitable reactive functional groups.
  • amplification of the molecule library consisting of the capture receptors can also be carried out.
  • this is a distortion-free Amplification method used in which the original composition of the molecule library is preserved.
  • An example of this is the emulsion PCR.
  • the functional groups intended for labeling may be introduced, for example, by using labeled amplification primers or labeled nucleoside triphosphate derivatives.
  • the catcher molecules are provided directly on the carrier, wherein - as described above - they can be coupled to the carrier via a cleavable bond.
  • the immobilized capture molecules are labeled as described above by introduction of one or more functional groups.
  • the immobilized labeled capture molecules are now brought into contact with the sample to be examined, wherein the target molecules contained in the sample bind to the immobilized capture molecules.
  • the remaining sample components may be, e.g. by rinsing the carrier, to be separated.
  • complexes of the capture molecules and the target sequences are detached from the carrier, from which the target molecules can be isolated.
  • contacting the capture molecules immobilized on the support with the sample may be under conditions of variable stringency, eg stringent or less stringent hybridization conditions, such that target molecules having weaker interactions with the immobilized capture molecules (eg nucleic acid sequences having one or more mismatches) , depending on the setting of the stringency bind to the capture molecules or not.
  • the separation of undesired sample components can be controlled by setting more or less stringent washing conditions.
  • the stringency can be controlled by temperature and / or buffer composition.
  • the functional groups with which the catcher molecules in the are labeled according to the invention preferably solid phase binding groups, ie groups which can be bound to a suitable solid phase, for example to another carrier as indicated above, a microtiter plate, a column or particulate solid phases such as spheres.
  • the functional group used is preferably a first member of a bioaffinity binding pair which can react with high affinity and specificity with the complementary second partner of the binding pair.
  • suitable pairs of binding partners are antigen or hapten / antibody, biotin / streptavidin, sugar / lectin, ligand / receptor, etc.
  • the functional group is biotin and the complementary group of the solid phase is streptavidin or avidin.
  • biotin derivatives which are capable of binding with streptavidin or avidin instead of biotin.
  • the target molecules bound to the capture molecules can be separated from other sample components, for example components of the sample that can not be bound with the capture molecules, in the method according to the invention.
  • the immobilization of the capture molecules on the solid phase can be location-specific, but also - when using different functionalized groups - also site-specific, e.g. on different particles or in different spatial areas of a common solid phase.
  • sub-fractions of the molecule library of capture molecules used to isolate the target molecules are prepared. These subfractions, which are preferably capable of binding with different species or groups of target molecules, can be prepared on a support spatially separated from other subfractions and spatially or / and temporally detached from / / copied from / to other subfractions from the support. Alternatively, sub-fractions of the molecule library can also be prepared separately on multiple supports. If necessary, different Subfractions of the molecule library can also be labeled with different functional groups. For example, a first subfraction of capture molecules can be labeled with biotin and bind to streptavidin or avidin-coated solid phase. Another subfraction may in turn be labeled with an antigen or hapten and bind to a solid phase coated with the appropriate antibody directed against the antigen or hapten.
  • Isolation of the target molecules preferably comprises elution of the target molecules from the solid phase under conditions in which the binding of the target molecule to the capture molecule is resolved, but the capture molecule remains bound to the solid phase.
  • the elution of the target molecules immobilized on the solid phase can take place in a single step.
  • a location- or / and time-specific elution can be carried out, wherein individual target molecules or individual groups of target molecules are eluted first in a first step of the solid phase and the elution of other target molecules or
  • Groups of target molecules in one or more subsequent steps takes place.
  • the elution is carried out by means of a temperature change, e.g. a temperature increase that leads to a denaturation of nucleic acid double strands.
  • the method according to the invention can also be used for the isolation of proteins and other molecules, in particular DNA-binding molecules, if capture probes suitable as catcher molecules are selected.
  • DNA-binding proteins can therefore be isolated with the aid of DNA capture probes, which can be in the form of a double or single strand.
  • peptide capture probes may also be used for the isolation of proteins or DNA molecules be used.
  • the capture probes used are nucleic acids having specific binding properties, for example aptamers or ribozymes.
  • the isolated target molecules can be used directly or indirectly for diagnostic or therapeutic purposes.
  • the extracted material can be used in follow-up reactions.
  • from extracted nucleic acids, proteins or peptides e.g. by transfer into suitable vectors (cloning), or into suitable target cells (transformation or transfection) or by in vitro translation, in particular isolation of mRNA target molecules.
  • follow-up reactions to which the isolated target molecules can be subjected are, in particular with nucleic acid target molecules, sequencing reactions or microarray analyzes.
  • nucleic acid fragments to be isolated / extracted from the support via suitable reagents and suitable temperatures (see FIG. 1G); 13. Further use of the selectively extracted nucleic acids, e.g. in a sequencing, PCR, cloning, microarray experiments.
  • the sample to be tested may be pretreated to selectively remove certain components from a sample containing a target molecule or a target molecule mixture.
  • Disturbing components may be removed, eg, repetitive elements or telomeric sequences upon analysis of gene fragments; Filtering out certain genes (eg housekeeping genes) in transcriptional analyzes; Protein or Protein classes that interfere with the proteome analysis of (rare) proteins.
  • known components can be trapped by binding to specific ("negative") catcher molecules specific for these interfering complements and only desired, eg known or unknown, target molecules eluted, resulting in a concentration of desired nucleic acids or proteins or other desired biomolecules for example, by a method comprising the steps of:
  • Positions are immobilized on or in the support, and (ii) passing a sample containing target molecules to be isolated through or over the support under conditions where interfering components from a sample can specifically bind to the capture molecule immobilized on the support.
  • interfering components are depleted so that subsequent analysis of the desired molecules can be performed with higher precision. Furthermore, a concentration of the desired target molecules of the sample to be examined is achieved and interfering molecules, e.g. several species in parallel, can be selectively removed from the molecule mixture.
  • the present invention also encompasses an embodiment relating to a method for isolating target molecules from a sample, wherein at least one process cycle in which the target molecules bind to immobilized capture molecules on a support, as in
  • WO 03/031965 and at least one process cycle in which the target molecules bind to free labeled capture molecules are combined.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (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)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

L'invention concerne un procédé d'extraction permettant l'isolement de molécules cibles contenues dans un échantillon au moyen d'une bibliothèque de molécules.
EP08851271A 2007-11-23 2008-11-24 Procédé d'extraction flexible permettant la production de bibliothèques de molécules spécifiques d'une séquence Withdrawn EP2217364A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007056398A DE102007056398A1 (de) 2007-11-23 2007-11-23 Flexibles Extraktionsverfahren für die Herstellung sequenzspezifischer Molekülbibliotheken
PCT/EP2008/009951 WO2009065620A2 (fr) 2007-11-23 2008-11-24 Procédé d'extraction flexible permettant la production de bibliothèques de molécules spécifiques d'une séquence

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EP2217364A2 true EP2217364A2 (fr) 2010-08-18

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US (1) US20100256012A1 (fr)
EP (1) EP2217364A2 (fr)
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US20100256012A1 (en) 2010-10-07
DE102007056398A1 (de) 2009-05-28

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