+

WO2009106322A1 - Procédé d'enrichissement ou d'analyse de fragments d'adn d'un échantillon d'adn génomique complexe - Google Patents

Procédé d'enrichissement ou d'analyse de fragments d'adn d'un échantillon d'adn génomique complexe Download PDF

Info

Publication number
WO2009106322A1
WO2009106322A1 PCT/EP2009/001362 EP2009001362W WO2009106322A1 WO 2009106322 A1 WO2009106322 A1 WO 2009106322A1 EP 2009001362 W EP2009001362 W EP 2009001362W WO 2009106322 A1 WO2009106322 A1 WO 2009106322A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
genomic dna
probes
fragments
carrier
Prior art date
Application number
PCT/EP2009/001362
Other languages
German (de)
English (en)
Inventor
Claudia Bauer
Peter Bauer
Olaf Riess
Original Assignee
Eberhard-Karls-Universitaet Tuebingen
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 Eberhard-Karls-Universitaet Tuebingen filed Critical Eberhard-Karls-Universitaet Tuebingen
Publication of WO2009106322A1 publication Critical patent/WO2009106322A1/fr

Links

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 enriching contiguous genomic DNA fragments from complex genomic DNA to be examined, and to a method for analyzing the DNA fragments obtained by this enrichment method, and to the use of the method for studying mutations in genomic DNA, in particular mutations in genes associated with certain diseases and / or in the identification of genes associated with certain diseases.
  • a major goal of molecular diagnostic science is namely to identify with the available methods and techniques by means of a specific enrichment within the complex structure of the genomic DNA sequence sections or genes about which diseases can be diagnosed and possibly also treated specifically.
  • novel sequencing technologies high density parallel sequencing
  • complete bacterial genomes 100 Mb
  • de novo sequenced within three to five days at a time.
  • human genome, as well as the genomes of many other species exceeds the sequencing capacity of these technologies many times over, because deviations from the normal state have to be investigated independently 5 to 20 times before, for example, a pathological mutation can be detected with sufficient certainty.
  • the size of the mammalian genomes (up to 3 Gb) has the great disadvantage that careful screenings can not be performed with individual experiments.
  • PCR approaches that are used as a basis for the mutation search must first be tested, which is very time-consuming and leads to a high personnel outlay. Performing the PCR reactions also requires a great deal of time and money, since these must be carried out using comparable protocols in order to achieve reproducible and meaningful results, so that on the one hand these reactions must first be established, and on the other hand often after the establishment the reactions are required for the analysis of an individual.
  • mutation search in unknown disease genes today presents the science with great challenges.
  • the positional cloning technique was used to identify disease genes, leading to relevant medical breakthroughs. Beginning with the coupling analysis in large families with the subsequent sequencing of candidate genes, recurring circumscribed sections of the human genome are analyzed.
  • Candidate regions that is, regions where there is a likelihood of mutations that can cause disease, are usually around 500 kb to 10 Mb in size. Therefore, it would be desirable to develop a method by which to enrich and subsequently analyze DNA molecules of any patient of that particular candidate region.
  • this object is achieved by a method comprising the following steps:
  • step a) providing double-stranded DNA probes having sequences which correspond to sequences in the complex genomic DNA to be examined, b) immobilization of the double-stranded DNA probes from step a) to a carrier,
  • the present invention provides an enrichment method of smaller DNA fragments of complex genomic DNA to be examined, which is highly flexible and which allows DNA fragments of interest to be rapidly and specifically enriched, after which they can subsequently be reliably analyzed, for example by means of a in vitro amplification or direct sequencing.
  • the present invention provides a novel method of sample enrichment which can successfully and reproducibly reduce the complexity of genomic DNA.
  • DNA probes are initially provided which are immobilized on a support, for example via specific markings.
  • the DNA probes, or the source of the probes are thereby selected specifically with regard to the genome section to be examined, and a random fragment length of about 100 bp to 3000 bp, in particular of between 250 bp and 1000 bp, can be found to be advantageous to have.
  • After immobilization of the probes they are denatured, ie the double strand of the DNA probes is broken up into two single strands.
  • the genomic DNA to be examined is randomly fragmented, with fragments having a size of approximately 100 to 3000 bp, in particular from 250 to 1000 bp, having proven particularly advantageous.
  • these details of the fragment lengths are merely exemplary, which is why smaller or larger lengths can be used.
  • the genomic DNA is optionally denatured, ie broken down into single strands, this being done, for example, by heating the fragments in a buffer, preferably in a hybridization buffer, or else alkaline.
  • a buffer preferably in a hybridization buffer, or else alkaline.
  • denaturation is not always necessary since it is already present in single-stranded form. It will be apparent to those skilled in the art that, depending on the nature and form of the genomic DNA to be tested, it must or may not provide for a denaturation step.
  • the fragmentation of the genomic DNA can be carried out, for example, by means of one or more restriction enzymes, by ultrasonic sonication, by nebulization.
  • the fragmented and optionally denatured genomic DNA is then added to the immobilized on the support probe, for example.
  • an amount of 0.1-20 micrograms wherein an amount of 10 to 20 micrograms has proved to be advantageous, after which the approach is incubated for hybridization over a period of time.
  • two DNA single strands possessing complementary base sequences assemble into duplexes.
  • the hybridization conditions between probe and target ie the fragments
  • Conditions are regularly selected which are generally uniformly stable independently of the sequences specifically involved, with factors to be taken into account being the buffer, incubation time and incubation temperature.
  • a hybridization buffer which contains 5 ⁇ SSC (0.75 M NaCl, 75 mM Na citrate, pH 7 0.1% (w / v) N-lauryl sarcosine, 0.02% (W / V) SDS, 2%.
  • the reaction is then incubated, for example, for a period of 1 to 80 hours, preferably 60 hours.
  • the support may be washed, for example, over one to several, preferably three, washes with a wash buffer, thereby removing any non-hybridization-bound portions of the target DNA.
  • a wash buffer any washing buffer can be used which has no influence on the hybridized products, but with which unbound genomic DNA fragments can be washed off.
  • the complementary to the single-stranded DNA probes bound, also single-stranded DNA fragments (the “targets” or targets) of the genomic DNA are mobilized, for example.
  • alkaline lysis or by heat By “elution” is meant any method of action by which the targets are detached from the carrier - and thus from the probes immobilized on the carrier. It is understood that fragments obtained hereby may have to be neutralized in the solution for further analysis, in particular if alkaline lysis is used for the immobilization. It will be clear to the person skilled in the art which conditions he has to choose for which analysis in order to be able to carry them out. Thus, it is important in the method according to the invention that the double-stranded DNA probes have already been denatured and added to single-stranded DNA before addition of the target DNA.
  • genomic DNA or “genome” is understood to mean the entire genetic material of an organism.
  • the DNA (deoxyribonucleic acid) derived from the genetic material in the chromosome of a particular organism is the “genomic DNA”.
  • a “genomic library” or “genomic library DNA” is a collection of clones made from a set of randomly generated, overlapping DNA fragments representing the entire genome of an organism. This genomic DNA, also referred to herein as “library DNA”, is often already present in single stranded form.
  • genomic "DNA fragments” are to be understood here as meaning smaller sections of the total genome, which may have a length of about 50 to about 5000 bp, with the genome of, for example, mammals being up to 3 Gb in size can own.
  • genomic DNA not only the entire genomic DNA of an organism derived or derived from the genetic material in the chromosome of a particular organism can be understood, but also those in the form of a library or library DNA fragments representing the entire genome of an organism.
  • a genomic library For the preparation of a genomic library, the entire cellular DNA of an organism is broken down by restriction enzymes into fragments, which are then inserted and propagated in a suitable cloning vector, for example bacteriophages or cosmids. If only a part of all DNA regions found in the genome of an organism is contained in a gene library, it is called a subgenomic library.
  • probe as used herein is meant an oligonucleotide capable of binding in a base-specific manner with a complementary strand of nucleic acid.
  • oligonucleotide each nucleic acid
  • the length of the oligonucleotides is usually at least 5, 10 or 20 bases long and can be up to 50, 100, 1000 or more bases long. Oligonucleotides may also include peptide nucleic acids or analogous nucleic acids.
  • a “carrier” is to be understood as meaning any substrate to which nucleic acid probes can be attached.
  • a plurality of different probes may be coupled to the surface of the carrier at various known locations.
  • Such carriers are then also referred to as “chips” or “microarrays”.
  • the surface of the carrier or the substrate can be produced in virtually any shape or in a plurality of surfaces, so that, for example, carriers in the form of (microtiter) plates, a sheet, of fibers or glass, etc. can be used, which is suitable for attaching probes.
  • the genome or genomic DNA to be tested is not limited to humans as a source, but extends to include, but is not limited to, mammals, plants, bacteria or cells derived from any of the above. It goes without saying that it is also possible to resort to "library DNA" as starting material for genomic DNA.
  • BAC clones bacterial artificial chromosome ", artificial bacterial chromosome
  • the freely available BAC cloning collection 32k (Osoegawa et al., "A Bacterial Artificial Chromosomal Library for Sequencing the Complete Human Genome", Genome Research 2001; 11: 483-496) is useful in the 150,000 to 250,000 contiguous bases of the human genome are each contained in an artificial bacterial chromosome, which can be produced at any time highly pure by bacteria.
  • fragmentation can be divided into smaller sections by means of one or more restriction enzymes, by ultrasound, by nebulization (compressed air or nitrogen).
  • the probes in step b) are immobilized on the support via a marking.
  • a marking means any chemical, biological or physical modification of the probes which make it possible to attach the probes to the support and thus immobilize it thereon.
  • a marker is used for labeling the double-stranded DNA probes, which has a specific or unspecific affinity for the carrier.
  • the probes bind to the carrier arbitrarily - via the marker - ie it does not depend on the nature of the surface of the carrier to which the probes are to be bound .
  • the probes bind very specifically to the - possibly correspondingly processed - surface of the carrier and possibly to specific locations on the latter. It is preferred for a specific affinity of the marker if the marker is biotin-dUTP. This can then, in a preferred development of the method, be attached terminally to the probe, again being preferred if a terminal transferase is used for the label.
  • Terminal transferase is an enzyme that mediates the template-independent attachment of deoxynucleotides (2'-deoxyribonucleoside monophosphates) to the terminal 3'-OH groups of DNA molecules. Therefore, this enzyme may, for example, attach biotin-dUTP to the DNA probes.
  • the carrier has a coating with a specific affinity for the marker, wherein, when the marker is biotin-dUTP, it is preferred if the immobilization of the labeled double-stranded DNA probes in step c) via streptavidin takes place, with which the carrier is coated.
  • Streptavidin a protein isolated from a bacterium, binds biotin to its four subunits. In general, however, any modification can be used which by high-affinity interactions between the partners of a specific binding pair, such. Biotin / streptavidin or avidin, hapten / anti-hapten antibody, sugar / lectin, etc., can be mediated.
  • the method is furthermore generally preferred if the carrier is selected from the group consisting of microtiter plates, centrifugation columns, magnetic beads, paramagnetic beads. It will be understood that any substrate that can be modified and used for the purposes herein can serve as a carrier.
  • the prior art discloses a multiplicity of carrier substrates which are suitable for the present method and which are well known to the person skilled in the art.
  • step d) is carried out before or in parallel with one of the previous steps. It is understandably preferred to accelerate the process if the step of fragmenting and denaturing the genomic DNA is carried out in parallel to the fragmentation and optionally labeling and immobilization of the probes. This However, it also depends on the probes / genomic DNA material to be used, and it will be clear to the person skilled in the art which sequence should be used in each case.
  • step c) and / or the genomic DNA in step d) by heat, alkali or acid and hydrogen bonding solvents, such as. As urea, formamide, is performed. It is understood that the skilled worker can also use denaturing agents in a mixture.
  • the elution of the genomic DNA fragments is carried out by methods selected from the group consisting of heat exposure, acid or alkali treatment and the use of hydrogen bonding solvents, such as. As urea, formamide.
  • the invention further relates to a method of analyzing genomic DNA characterized by the steps of enrichment by the method of the invention and analyzing the eluted genomic DNA fragments.
  • the analysis is carried out by one or more of the methods selected from the group consisting of polymerase chain reaction, sequencing, in particular direct sequencing, and clonal amplification on glass surfaces.
  • the PCR which amplifies specific DNA fragments in vitro, is a highly sensitive technique that allows one or more specific double-stranded DNA fragments to accumulate millions of times within a very short time, even from very small amounts of heterogeneous DNA.
  • the so-called emPCR emulsion based PCR
  • the microbeads are coupled to the single-stranded DNA fragments, with the amplification reagents in emulsified in an oil-water mixture (see, for example, Margulies et al., (2005) Nature; 437: 376-380).
  • the genomic DNA obtained by enrichment can also be sequenced, for example, with so-called next-generation sequencing machines in which, instead of cloning into bacterial or viral systems for propagation of individual sequences, a direct clonal amplification of individual molecules takes place.
  • the genomic DNA is provided after the fragmentation in a further step with PCR or Sequenzieradaptern (production of the so-called library).
  • the recovered library DNA can then be amplified directly clonally (for example, microbead-coupled emPCR (Margulies et al. (2005) Nature; 437: 376-380) for the 454 method (see www.
  • the method be used to study mutations in genomic DNA, wherein in one embodiment it is preferred that the method be used to study mutations in genes associated with certain diseases, and preferred in another embodiment when the method is used to identify genes associated with certain diseases.
  • LRRK2 mutations in genes known to be associated with certain diseases or symptoms are investigated. For example, it has recently been shown that a single gene mutation is the cause of one of 25 Parkinson's cases worldwide. Leave the studies suggest that the mutation in a recently discovered gene called LRRK2 causes about five percent of inherited Parkinson's cases and about two percent of sporadic cases.
  • hitherto unknown genes can be identified with the method according to the invention, which are associated with diseases.
  • risk genes for Crohn's disease and type 2 diabetes have recently been identified.
  • the methods of the present invention provide an excellent tool to improve such analyzes, both in terms of cost and time required, and in reliability and flexibility.
  • FIG. 1 Genomic enrichment of human DNA sections complementary to chromosome 5-BAC (RPH 795P7).
  • a fragmented and biotin-labeled BAC probe (RP11795P7, chromosome 5) was used, at a concentration of 150 ng / ⁇ l. Of these, 1.0 .mu.l, 0.5 .mu.l or 1.0 .mu.l of a 1:10 dilution were used. The average length of the fragments was about ⁇ 400 bp (base pairs). Fragmentation was performed by nebulizing the BAC DNA to 200 to 800 bp fragments. This was followed by purification of the fragments over a MinElute ® column (Qiagen, Hilden, Germany). The fragments were further labeled with biotin by a terminal transferase with biotin-16-ddUTP (each Roche Diagnostics, Mannheim, Germany).
  • human genomic DNA was fragmented by nebulization to about 200 bp to 800 bp. These DNA fragments were purified by MinElute ® column and small fragments ( ⁇ 250 bp) by Ampure ® -Beads (® Agenco ⁇ rt Bioscien- ce, Beverly MA, USA) away. After elution of the DNA from the beads, the fragments were "end-poled” and phosphorylated by T4 DNA polymerase and T4 polynucleotide kinase (PNK) and dATP (New England Biolabs GmbH, Frankfurt, Germany), followed by further purification a MinElute ® column.
  • PNK T4 polynucleotide kinase
  • the fragments were used at a concentration c of 580 ng / ul to give 4.0 ul (equivalent to about 2 ug) 2.0 ul (corresponding to about 1 ug) and 1.0 ul (the length of the fragments averaged about 600 bp, pooled F # 17, and if the fragments were to be used for amplification after enrichment, for example, the "polis hed" DNA fragments specific adapter (single strand adapter or double strand adapter) ligated.
  • streptavidin-coated microtiter plates were used by Thermo Scientific (Waltham, USA, catalog number 95 029 362).
  • the biotin labeled BAC probe (150 ng, approximately 400 bp) was loaded with 50 ⁇ l binding buffer (10 mM Tris-HCl, 2M NaCl, ImM EDTA, 0.1% Tween 20, pH 7.6) into the wells of the streptavidin-coated ones Plate pipetted.
  • the wells were then sealed with adhesive film and incubated for 1 hour at room temperature in the Platten thoroughlyelthermoblock at speed 2. Subsequently, the plate was briefly centrifuged and the buffer removed; This was followed by a washing step with 0.5 M NaOH and a 10-minute incubation with 100 ⁇ l 0.5M NaOH to denature the immobilized probes.
  • hybridization buffer 5x SSC (0.75M NaCl, 75 mM NaCitrate, pH 7 0.1% (w / v) N-lauryl sarcosine, 0.02% (w / v) SDS, 2% Blocking Reagent ( 20 mg) and denatured for 5 minutes by boiling at 95 ° C.
  • 100 ⁇ l of hybridization buffer preheated to hybridization temperature were initially introduced, and the denatured DNA was pipetted in, followed by closed the wells with adhesive film.
  • the plate was incubated for about 60 hours at about 55 ° C on the thermoblock at speed 2 to achieve in the wells a temperature of about 50 0 C, at which the fragments hybridize to the probes.
  • the plate was tightly closed to prevent dehydration. Subsequently, the plate was briefly centrifuged. The wells were washed with yes 300 .mu.l PBS + 0.05% Tween 20 (and the contents of the wells mixed carefully twice with the pipette); the buffer was then removed and allowed to dry on paper for the last wash cycle in reverse.
  • the PCR detection was carried out using primers for the markers D5S818 (9791/9792) and D13S317 (9793/9794) in a multiplex reaction, the subsequent fragment analysis being carried out on the CEQ8000 (Beckman Coulter).
  • the results are shown in FIG. It can be seen that the BAC clone (chromosome 5) used for the enrichment showed the D5S818 allele "13".
  • the heterozygous human DNA used showed the D5S818 features "11” and "12” (Chromatogam D). All genomic enhancements were positive for the expected features "11” and "12” as well as for a more or less pronounced feature "13” (residues of BAC DNA in the eluate, Chromatogram AC).
  • the method according to the invention it was possible to achieve a 10,000-fold enrichment of the specific DNA sections with separation of the unspecific DNA.
  • a high enrichment efficiency offers the advantage that the enriched DNA can be used, for example, in an emulsion PCR (em-PCR) and in a subsequent GS-FLX sequencing.
  • em-PCR emulsion PCR
  • GS-FLX sequencing GS-FLX sequencing.
  • in the method according to the invention can be dispensed with a PCR as an enrichment method, thereby avoiding the often occurring in a PCR uncontrolled base changes, which would lead to a falsified result.

Landscapes

  • 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)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • 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

L'invention concerne un procédé d'enrichissement de petits fragments d'ADN d'un ADN génomique complexe, ainsi qu'un procédé d'analyse d'ADN génomique, le procédé étant utilisé pour l'enrichissement.
PCT/EP2009/001362 2008-02-29 2009-02-26 Procédé d'enrichissement ou d'analyse de fragments d'adn d'un échantillon d'adn génomique complexe WO2009106322A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008013715.4 2008-02-29
DE200810013715 DE102008013715B4 (de) 2008-02-29 2008-02-29 Verfahren zur DNA-Analyse

Publications (1)

Publication Number Publication Date
WO2009106322A1 true WO2009106322A1 (fr) 2009-09-03

Family

ID=40612940

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/001362 WO2009106322A1 (fr) 2008-02-29 2009-02-26 Procédé d'enrichissement ou d'analyse de fragments d'adn d'un échantillon d'adn génomique complexe

Country Status (2)

Country Link
DE (1) DE102008013715B4 (fr)
WO (1) WO2009106322A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2913660A4 (fr) * 2012-10-25 2016-06-29 Olympus Corp Méthode de détection de particules cibles
US9488578B2 (en) 2011-08-26 2016-11-08 Olympus Corporation Single particle detection device, single particle detection method, and computer program for single particle detection, using optical analysis
US9575060B2 (en) 2012-04-18 2017-02-21 Olympus Corporation Method for detecting a target particle
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0487104A1 (fr) * 1990-11-22 1992-05-27 Sumitomo Electric Industries, Ltd Méthode pour immobiliser sur un support de l'ADN simple-brin faisant réagir uniquement sa partie terminale
EP1184466A2 (fr) * 2000-08-26 2002-03-06 Affymetrix, Inc. Enrichissiment et amplification des cibles d'acide nucleique pour l'analyse dans un réseau
EP1630237A2 (fr) * 2004-08-26 2006-03-01 Agilent Technologies, Inc. Isolement d'acides nucléiques par hybridation avec une phase solide
US20070141604A1 (en) * 2005-11-15 2007-06-21 Gormley Niall A Method of target enrichment

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0487104A1 (fr) * 1990-11-22 1992-05-27 Sumitomo Electric Industries, Ltd Méthode pour immobiliser sur un support de l'ADN simple-brin faisant réagir uniquement sa partie terminale
EP1184466A2 (fr) * 2000-08-26 2002-03-06 Affymetrix, Inc. Enrichissiment et amplification des cibles d'acide nucleique pour l'analyse dans un réseau
EP1630237A2 (fr) * 2004-08-26 2006-03-01 Agilent Technologies, Inc. Isolement d'acides nucléiques par hybridation avec une phase solide
US20070141604A1 (en) * 2005-11-15 2007-06-21 Gormley Niall A Method of target enrichment

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALBERT THOMAS J ET AL: "Direct selection of human genomic loci by microarray hybridization", NATURE METHODS, NATURE PUBLISHING GROUP, GB, vol. 4, no. 11, 1 November 2007 (2007-11-01), pages 903 - 905, XP002499757, ISSN: 1548-7091, [retrieved on 20071014] *
BASHIARDES STAVROS ET AL: "Direct genomic selection", NATURE METHODS JAN 2005,, vol. 2, no. 1, 1 January 2005 (2005-01-01), pages 63 - 69, XP002525087 *
LOVETT ET AL: "Fishing for complements: finding genes by direct selection", TRENDS IN GENETICS, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 10, no. 10, 1 October 1994 (1994-10-01), pages 352 - 357, XP023477939, ISSN: 0168-9525, [retrieved on 19941001] *
OKOU DAVID T ET AL: "Microarray-based genomic selection for high-throughput resequencing", NATURE METHODS NOV 2007,, vol. 4, no. 11, 1 November 2007 (2007-11-01), pages 907 - 909, XP002524506 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9488578B2 (en) 2011-08-26 2016-11-08 Olympus Corporation Single particle detection device, single particle detection method, and computer program for single particle detection, using optical analysis
US9575060B2 (en) 2012-04-18 2017-02-21 Olympus Corporation Method for detecting a target particle
EP2913660A4 (fr) * 2012-10-25 2016-06-29 Olympus Corp Méthode de détection de particules cibles
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

Also Published As

Publication number Publication date
DE102008013715A1 (de) 2010-04-08
DE102008013715B4 (de) 2011-12-08

Similar Documents

Publication Publication Date Title
DE69433136T2 (de) Nukleinsäure-isolierung
DE69825240T2 (de) Verfahren zur subtraktiven hybridisierung und differenziellen analyse
US6156502A (en) Arbitrary sequence oligonucleotide fingerprinting
US20120045771A1 (en) Method for analysis of nucleic acid populations
DE69711895T2 (de) Characterisierung von dna
DE112010004821T5 (de) Prozessierung amplifizierter DNA-Fragmente zur Sequenzierung
US20070148690A1 (en) Analysis of gene expression profiles using sequential hybridization
DE112013004650T5 (de) Multiplexpyrosequenzierung unter Verwendung nichtinterferierender, rauschbeendender Polynukleotididentifikationstags
DE102008013715B4 (de) Verfahren zur DNA-Analyse
DE10149947A1 (de) Mikrofluidisches Extraktionsverfahren
DE102005034628B4 (de) Verfahren zur Untersuchung von Cytosin-Methylierungen in DNA
EP1366195A2 (fr) Procede de detection de molecules d'acide nucleique
KR20240085399A (ko) Dna 단편의 멀티 결합 조립 반응을 이용한 단일세포의 다중체 전장 시퀀싱 분석방법
DE102019118332B4 (de) Verfahren und testkit zur bisulfitmodifizierung von dna
EP0698122B1 (fr) Agent de diagnostic complexe de l'expression genetique et son procede d'application en diagnostic medical et pour isoler des genes
EP1816217B1 (fr) Méthode pour la détection d'une pluralité d'acides nucléiques cibles
US20100204050A1 (en) Target preparation for parallel sequencing of complex genomes
EP1537246B1 (fr) Procédé d'amplification d'informations génétiques utilisant des oligonucléotides se liant aux sites multiples dans le génome
US20210198807A1 (en) Methods and systems for production of dna libraries directly from a stool sample for 16s metagenomics next generation sequencing
DE10240746A1 (de) Verfahren zum Nachweis von Nukleinsäuresequenzen mittels spaltbarer Sondenmoleküle
EP2471955B1 (fr) Molécule d'acide nucléique hybridisable spécifique à l'organisme
EP2706124B1 (fr) Mise en évidence simultanée de différentes micro-formes de biogenèse RNA
DE19821114A1 (de) Methode zur Identifizierung und Darstellung interindividueller DNA-Sequenzunterschiede
RU2585519C2 (ru) Способ селективного отбора целевых участков днк
EP1650314A1 (fr) Détermination dynamique d'analytes en utilisant une puce localisée sur une surface interne

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09713694

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09713694

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载