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WO2009069843A1 - Procédés de fabrication de microréseaux de métagénomes et détection quantitative de gènes de micro-organismes au moyen de microréseaux de métagénomes - Google Patents

Procédés de fabrication de microréseaux de métagénomes et détection quantitative de gènes de micro-organismes au moyen de microréseaux de métagénomes Download PDF

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Publication number
WO2009069843A1
WO2009069843A1 PCT/KR2007/006645 KR2007006645W WO2009069843A1 WO 2009069843 A1 WO2009069843 A1 WO 2009069843A1 KR 2007006645 W KR2007006645 W KR 2007006645W WO 2009069843 A1 WO2009069843 A1 WO 2009069843A1
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Prior art keywords
microarray
metagenomes
group
quantitative detection
microorganisms
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English (en)
Inventor
Jin-Woo Bae
Young-Do Nam
Ho-Won Chang
Kyoung-Ho Kim
Seong Woon Roh
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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

Definitions

  • the present invention relates to a method for quantitative detection of genes or microorganisms in metagenomes by microarray prepared by arranging massive metagenomes at high density on a glass slide without any
  • DNA microarray facilitates successive analysis and is very powerful technique for efficient detection and quantitative detection of nucleic acid.
  • Microarray has been widely used in the fields of biological science including environmental microbiology and microbiological ecology, precisely in detection and diagnosis in human, animal, plant and foods.
  • DNA microarray is effective in explaining important matters happening in microbial ecosystem, it has a few practical problems such as low sensitivity and resolution delaying its general application (Cook et al., Curr Opin Biotechnol, 14, 311-318, 2003) .
  • the above limits have to be overcome.
  • the most representative microarray is oligomer chip targeting microbial 1 ⁇ 5S rRNA gene (50-70 oligomer chip), which is an advanced one facilitating detection of multiple uncultivated microorganisms in high efficiency but still having the problems of low sensitivity and resolution at species level.
  • GPM gene-probing microarray
  • GPM was established based on a concept of big genomic difference among different microbial species (hybridization is not happening in between) , which provided high resolution and overcame the problem of the conventional oligomer chip, the low sensitivity (Bae et al . , Appl Environ Microbiol, 71, 8825-8835, 2005) .
  • GPM was the firstly introduced microarray format for the ecological analysis of microorganisms in environment, which was high efficient diagnostic method of bioprocess (Bae, J-W. et al., Appl Environe Microbiol., Vol.71, No.12, 8825-8835, 2005).
  • microarray simply constructed by direct printing of subspecies specific genomes only, based on the fractionation of unique genome of each microorganism by SSH (substrate suppression hybridization), could be effective in accurate detection of microorganism subspecies or lower (Bae et al., Nucleic Acids Res. 33, ell3, 2005, Korean Patent No. 10-0758374) .
  • SSH substrate suppression hybridization
  • the biggest disadvantage of this genome direct printing microarray is that only cultivated microorganism genomes can be used. Therefore, genomes of those at least 99% of microorganism, which are uncultivated, are useless, suggesting that most of microorganisms are undetectable.
  • the present inventors investigated whether gene or microorganism specific sequence probe could be precisely detected in the microarray constructed by printing tens - tens of thousands of metagenomes extracted from the nature. And the present inventors completed this invention by confirming that various species of included in different metagenomes could be detected by one time detection using this microarray.
  • the present invention provides a microarray in which metagenomes extracted from environment or their fragments are printed on the solid support .
  • the present invention also provides a method for producing the microarray for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the following steps:
  • step 2) printing the metagenomes purified in step 2) on the solid support.
  • the present invention further provides a method for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the following steps: 1) labeling DNA separated from the samples;
  • the present invention provides a kit for quantitative detection of specific genes or microorganisms included in metagenomes comprising the microarray.
  • the present invention relates to a method for quantitative detection of genes or microorganisms living in nature by using the microarray prepared by arranging massive metagenomes extracted from environment on a glass slide at high density, without any PCR.
  • the method of the present invention is improved from the conventional method for detecting a microorganism or diagnostic DNA chip requiring all the processes of labeling of each and every sample, hybridization, scanning and data analysis. So, the method of the invention facilitates quantitative detection of microorganisms or genes of massive metagenomes with one DNA chip by one time detection.
  • Fig. 1 is a schematic diagram illustrating the construction process of the metagenome microarray in which metagenomes extracted from environment were arranged on a glass slide.
  • Fig. 2 is a diagram illustrating that when a specific metagenome was hybridized with the metagenome microarray, the homologous metagenome among the arranged metagenomes on the slide showed the highest hybridization signal.
  • Fig. 3 is a graph illustrating the quantitative expression of the results of Fig. 2.
  • Fig. 4 is a diagram illustrating the detection of a specific microorganism on the microarray where 16S rDNA genes were printed, without additional amplification process :
  • A is a diagram illustrating the limitation of the conventional sequencing probe.
  • the conventional E. coli specific sequencing probe comprising 17 mer was constructed. The end of the probe was treated with cy-5, followed by hybridization with the microarray. E. coli 16S rDNA was detected, but E. coli genome was not detected;
  • B is a diagram illustrates that when the end of the E. coli specific sequencing probe was treated with biotin and the probe was hybridized by TSA (tyrimide signal amplification) , E. coli 16S rDNA and its genome were both detected;
  • C is a diagram illustrating the Bacteroides group specific detection by TSA using the Bacteroides-biotin sequencing probe.
  • D is a diagram illustrating the detection of the whole microorganism on the 16S rDNA microarray using the microorganism sequencing probe constructed for the detection of the whole microorganism.
  • Fig. 5 is a diagram illustrating the detections on the metagenome microarray each using sequence probe for the detection of the whole microorganism, sequence probe for the detection of Bacteroides group, sequence probe for the detection of Firmicutes and sequence probe for the detection of E. coli.
  • Fig. 6 is a graph illustrating the quantitative expression of the results of Fig. 5.
  • Fig. 7 is a diagram illustrating the detection of the whole 16S rDNA genes of metagenomes on the metagenome microarray after labeling them with photobiotin and amplifying the signals by TSA.
  • “metagenome” indicates the total DNA extracted from various environments on earth including water, sea, soil, air, foods, waste water or intestines or tissues of animal (including human) and plants.
  • microarray indicates the first or secondary structural array having separated sections divided regularly on the solid support.
  • microarray means bio-chip or DNA chip wherein thousands or tens of thousands of nucleic acids or proteins are arranged at regular intervals on the solid support to which a target substance is treated, and the binding pattern is investigated.
  • printing indicates the fixation of metagenomes extracted from the natural environment on the microarray, which is also called “spotting”.
  • labeling indicates labeling for detecting the hybridization of a target DNA with the microarray probe.
  • hybridization indicates coupling of complementary nucleic acids.
  • the degree of hybridization is determined by the level of complementation, Tm of the generated hybrids, stringency of reaction conditions or GC content of nucleic acid.
  • the present invention also provides a microarray in which metagenomes extracted from environment or their fragments are printed on the solid support.
  • the present invention also provides a method for producing the microarray for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the following steps:
  • step 2) printing the metagenomes purified in step 2) on the solid support.
  • the metagenomes of step 1) are preferably extracted from one or more places selected from the group consisting of water, sea, soil, air, foods, waste water, intestines or tissues of animals including human and plants, but not always limited thereto.
  • the present inventors collected human fecal samples and further extracted metagenomes therefrom using extraction buffer and phenol-chloroform.
  • the purification of step 2) is preferably performed by ethanol precipitation or using DNA separation kit, but not always limited thereto.
  • the purified metagenome can be used as it is or additionally partialized or fragmented before use.
  • the partialization is performed by SSH (Suppression Subtractive Hybridization) , and fragmentation is performed using BAC library (bacterial artificial chromosome library) or fosmid library, but not always limited thereto.
  • SSH Selection Subtractive Hybridization
  • BAC library bacterial artificial chromosome library
  • fosmid library bacterial artificial chromosome library
  • SSH suppression subtractive hybridization
  • SSH is the widely used method for selecting DNA molecule distinguishing two DNA libraries closely related (Rebrikov et al., Methods MoI Biol., 258, 107-134, 2004) .
  • the possible application of SSH to metagenome library was proposed.
  • the importance of this method is that the method facilitates normalization and subtraction of genes existing in two different metagenome libraries. Normalization indicates the equalization of many DNA fragments in a target group, while subtraction indicates elimination of the overlapping nucleotide sequence between two groups. Particularly, DNAs are extracted from the two groups and the extracted DNAs are digested with proper restriction enzymes.
  • the DNA fragments are hybridized by using an adaptor specific to DNA fragment specifically existed in metagenome.
  • Mriver' the strain for comparison
  • ⁇ tester' the strain comprising specific DNA which dose not belong to driver
  • PCR is performed to amplify tester-specific fragments. That is, DNA fragments existing in tester metagenome are selectively amplified and in the meantime, other DNA fragments existing in driver are not amplified.
  • SSH facilitates the identification of a gene, particularly it enables the detection of slight difference in genes of two very close strains by amplifying a specific target region of a gene, and favors the understanding of genetic diversity in metagenomes obtained from environment.
  • a specific metagenome to be used for comparison is used as a SSH driver, and a sample metagenome is used as a SSH tester.
  • Tester and driver samples are digested with Rsa I in equal fragment length, and 100-1500 bp DNA fragments are confirmed on agarose gel by staining with ethidium bromide.
  • SSH can be preferably performed by the protocol of PCRselect Bacterial Genome Subtraction Kit (Clontech) , but not always limited thereto.
  • the conventional fosmid library or BAC library is preferably used but not always limited thereto.
  • metagenomes are firstly extracted from environments existing millions of various microorganisms, for example soil, sea water, tidal flat, river, animal intestines, etc. Then, the extracted metagenome is cloned into a vector.
  • the conventional plasmid can be used, but BAC, YAC, fosmid and cosmid which are capable of cloning the bigger gene or gene cluster are preferred.
  • BAC Bacterial Artificial Chromosome
  • fosmid are more preferred.
  • BAC vector is very useful for the analysis of 10 - 300 kb sized big DNA fragment, so that it has been used for human genome project and animal/plant (rat and rice) genome analysis.
  • Fosmid can allow approximately 37-52 kb sized gene or genome in and has high transformation efficiency.
  • the fragmented metagenomes recombinated by being introduced into a vector form a library by being preserved and expressed in culturable microorganisms.
  • E. coli As a host cell for expression thereof, E. coli has been widely used. However, when Gram-negative E. coli is used alone, not every random metagenome gene can be expressed. Therefore, other host cells such as Bacillus or Streptomyces and proper vectors for the cloning and expression in the host cells can be also used.
  • the printing of step 3) is preferably performed by microspotting using a pin, microdropping based on inkjet principle or addressing using electricity and when it is performed by pin microarray, a pen having a slit like quill of the end at the pin is preferably used, but not always limited thereto.
  • the present inventors constructed a minimized microarray with high reproducibility by quadruple spotting of metagenome DNA using a microarray machine.
  • the printing herein is to arrange tens-tens of thousands of metagenomes on a solid support by the method well informed to those in the art, but not always limited thereto. In this invention, 216 metagenomes were printed on a glass slide.
  • the solid support of step 3) is preferably slide glass, silicon chip, nitrocellulose or nylon membrane, but not always limited thereto. And any support that is usable for hybridization can be accepted.
  • the surface of the support is not limited as long as single stranded or double stranded nucleic acid can be fixed by covalent bond or non-covalent bond thereon.
  • the support having hydrophilic or hydrophobic functional group such as hydroxyl group, amino acid, thiol group, aldehyde group, carboxyl group and acyl group on its surface is preferably used, but not always limited thereto.
  • the surface of the support itself is preferably the glass treated with a silane coupling agent on the market such as amino alkylsilane, or treated with polycation such as polylysine and polyethyleneamine, but not always limited thereto.
  • a silane coupling agent on the market such as amino alkylsilane
  • polycation such as polylysine and polyethyleneamine, but not always limited thereto.
  • the present inventors performed spotting on a slide glass, and cross-linking by UV irradiation for the fixation of the probe. As a result, the support durable under proper humidity for a long while was prepared.
  • the present invention further provides a method for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the following steps:
  • the sample of step 1) is preferably obtained from environment, human intestines, animal intestines, water or foods, or artificially synthesized oligonucleotides but not always limited thereto.
  • the labeling of step 1) is preferably performed by using one of fluorescent materials selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC) and rhodamine, but not always limited thereto.
  • DNA labeling is performed as follows; the gene fragment (1 kb or up to 20 b in size) of a target microorganism is labeled with photobiotin, which is conjugated with streptavidin-tyrimide-Cy5. This labeling demonstrates signal amplification effect 10 times at least and 50 - 100 times the effect of the conventional direct labeling using Cy5.
  • the signal amplification effect by biotin-tyrimide-Cy5 is 5 - 10 times the amplification effect of the conventional Cy5
  • the signal amplification effect by photobiotin-tyrimide-Cy5 using photobiotin is 5 - 10 times the effect by single biotin-tyrimide-Cy5. Therefore, the signal amplification effect by the labeling of the invention is 50 - 100 times the effect of the conventional method using Cy5 as a whole.
  • the method of the invention is also compared with the biotin-dUTP labeling. According to the conventional labeling using biotin-dUTP, the total amplification is ambiguous and the amount of oligomer actually used for hybridization can not be confirmed.
  • the present inventors constructed E. coli specific sequence probe and treated its end with Cy5 and then hybridized with the microarray. As a result, 16S rDNA of E. coli was detected but the genome was not detected. However, when the probe was treated with photobiotin-TSA-Cy5 and hybridized with the microarray, not only 16S rDNA of E. coli but also the genome was detected. Therefore, it was confirmed that photobiotin-tyrimide-Cy5 based labeling was much more effective to increase the signal of the sequence probe to be hybridized with the microarray than the conventional Cy5 based labeling (see Fig. 7) .
  • the process of hybridization and washing of step 2) can be performed by the conventional method under proper conditions with regulating a denaturant, temperature and concentration of salts.
  • formamide or dimethyl sulfoxide is used as a denaturant included in hybridization buffer.
  • concentration of a denaturant is preferably included by 10- 70% and more preferably included by 30-50 %, but not always limited thereto.
  • temperature for hybridization and concentration of salts in washing solution can be regulated.
  • the concentration of salts in washing solution is preferably 0-lx SSC, and more preferably 0.01-0. Ix SSC, but not always limited thereto.
  • the detection of step 3) can be differently executed according to the method of DNA labeling.
  • the microarray is washed after hybridization in order to eliminate those genes not- hybridized, and then, the hybridized genes were investigated using high-resolution fluorescence scanner such as laser fluorimeter to identify uncultivated target microorganism, but not always limited thereto.
  • the laser- induced fluorescence using fluorescent dyes is widely used these days, which overcomes the detection limit of fluorescence and has advantages of less background noise.
  • CCD or confocal laser in light launching part can be used. Nucleic acid derived from the sample can be labeled by Cy3 or Cy5.
  • absorption wavelength of Cy3 is 550 nm and emission wavelength is 570 nm.
  • Absorption wavelength of Cy5 is 649 nm and emission wavelength is 670 nm. Both wavelengths express different colors, green and red, which favors comparison.
  • the degree of fluorescence can be measured quantitatively by representing the fluorescence by numbers .
  • the results of hybridization with the microarray on the slide glass are changed into video images by fluorescence scanner.
  • the video images are outputted from the scanner by BMP or TIFF.
  • Image processing software representing the fluorescence signal of each spot as numbers is equipped to the scanner or separately used.
  • the conventional DNA chip constructed as a microarray using probes for the detection of specific genes and microorganisms requires hybridization of massive samples with the equal amount of metagenomes.
  • the detection method of the present invention does not require hybridization of samples with each corresponding metagenome, and instead the method requires only simple hybridization with small amount of sequence probe for the detection of specific genes and microorganisms. Therefore, the present invention provides a method for quantitative detection of microorganisms existing in metamaterials extracted from environment by treating microorganism specific or gene specific sequence probe on the metagenome microarray constructed by printing those metamaterials directly on the microarray.
  • the present invention also provides a kit for quantitative detection of specific genes or microorganisms included in metagenomes comprising the microarray.
  • the conventional DNA chip for the diagnosis of microorganisms or genes has disadvantage of complicated processes of a whole bunch of labeling for every sample, hybridization, scanning and data analysis.
  • the kit using the microarray of the present invention simplifies the detection of genes or microorganisms without any additional amplification process by high-density printing of metagenomes extracted from environment samples directly on the microarray as one sample.
  • Example 1 Construction of a microarray with metagenomes of human intestine microorganisms
  • the present inventors extracted metagenomes from human fecal samples.
  • the fecal samples were taken from 8 volunteers over 6 times and stored at -80 ° C. The samples were pulverized in a mortar along with thawing-freezing repeatedly using liquid nitrogen until they turned into fine powders.
  • DNA was extracted from cell walls of microorganisms in the samples by using extraction buffer containing SDS and tris-EDTA.
  • Metagenomes of the fecal samples were extracted using phenol-chloroform and purified by ethanol precipitation. Metagenomes were additionally purified by using MOBIO (Mo Bio Laboratories, Solana Beach, CA) DNA isolation kit.
  • MOBIO Mo Bio Laboratories, Solana Beach, CA
  • the metagenome extracted from the human fecal sample was concentrated to the final concentration of 400 ng/ul by using SpeedVac (Low Vacuum SpeedVacSystems, Telechem
  • Example 2 Labeling of metagenome and microorganism group specific nucleotide (sequence probe) and hybridization
  • the present inventors performed labeling as follows.
  • Nucleotide sequences of the total 16S ribosomal gene of bacteria, E. coli group, Firmicutes group, and Bacteroides group were obtained from NCBI (National Center for Biotechnology Information) nucleotide sequence database, and sequence probe for each group was constructed (Table 2) .
  • Microorganism specific nucleotide was treated with the mixed solution comprising 500 ul of TNB buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent) , 300 ul of streptavidin-HRP (PerkinElmer Life science, Inc. U. S. A) and 300 ul of tyrimide-Cy5 (PerkinElmer Life science, Inc. U. S. A) for the secondary tyrimide-Cy5 reaction, after washing the slide.
  • TNB buffer 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent
  • streptavidin-HRP PerkinElmer Life science, Inc. U. S. A
  • tyrimide-Cy5 PerkinElmer Life science, Inc. U. S. A
  • the slide was washed three times with TNT buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) and then washed again with O.O ⁇ x SSC solution.
  • TNT buffer 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20
  • O.O ⁇ x SSC solution was dried by using a centrifuge .
  • Example 3 Microarray scanning and data analysis
  • A2 - A6 probes also exhibited strong signals, even though there was a slight margin (30 - 78%) .
  • Bl - Fl samples exhibited 100% signal of their own and their comparative groups showed significantly high signals.
  • their comparative groups G2 - G6, H2 - H ⁇

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Abstract

La présente invention concerne un procédé de détection quantitative de gènes ou de micro-organismes au moyen d'un microréseau préparé en plaçant des quantités massives de métagénomes sur une lame de verre à haute densité, plus précisément un procédé de détection quantitative de gènes ou de micro-organismes au moyen d'un microréseau sur lequel des métagénomes, l'ADN total obtenu à partir d'échantillons environnementaux ont été imprimés à haute densité après purification, fragmentation et séparation. Selon le procédé de la présente invention, des dizaines de milliers à des centaines de milliers d'échantillons peuvent être analysées quantitativement en même temps sans processus d'amplification en utilisant une sonde appropriée. Malgré son excellente spécificité, le procédé de détection traditionnel est limité en termes de détection, car il ne peut analyser qu'un échantillon à la fois, ce qui signifie que pour analyser un grand nombre d'échantillons, il faut répéter l'analyse de nombreuses fois. Le procédé de la présente invention résout le problème du procédé traditionnel et facilite ainsi la détection quantitative de gènes (ou de groupes de gènes) ou de micro-organismes (ou de groupes de micro-organismes), y compris par dizaines de milliers à centaines de milliers d'échantillons en même temps avec précision.
PCT/KR2007/006645 2007-11-30 2007-12-18 Procédés de fabrication de microréseaux de métagénomes et détection quantitative de gènes de micro-organismes au moyen de microréseaux de métagénomes Ceased WO2009069843A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170022573A1 (en) * 2014-04-11 2017-01-26 The Trustees Of The University Of Pennsylvania Compositions and Methods for Metagenome Biomarker Detection
CN111455021A (zh) * 2019-01-18 2020-07-28 广州微远基因科技有限公司 去除宏基因组中宿主dna的方法及试剂盒
EP4299713A4 (fr) * 2021-02-26 2025-01-22 Yokogawa Electric Corporation Procédé de mesure et système de mesure

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HYUN JU CHOI ET AL.: "Micropatterning of biomolecules on glass surfaces modified with various functional groups using photoactivatable biotin", ANALYTICAL BIOCHEMISTRY, vol. 347, 2005, pages 60 - 66 *
JIN-WOO BAE ET AL.: "Homogeneous versus heterogeneous probes for microbial ecological microarray", TRENDS IN BIOTECHNOLOGY, vol. 24, no. 7, July 2006 (2006-07-01), pages 318 - 323 *
JONATHAN L. ET AL.: "Metagenomic profiling: microarray analysis of an environmental genomic library", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 69, no. 8, August 2003 (2003-08-01), pages 4927 - 4934 *
LIXIN ZHANG ET AL.: "Library on a slide for bacterial comparative genomics", BMC MICROBIOLOGY, vol. 4, 22 March 2004 (2004-03-22), pages 12, XP021002560, DOI: doi:10.1186/1471-2180-4-12 *
SOO-JE PARK ET AL.: "Metagenome microarray for screening offosmid clones containing specific genes", FEMS MICROBIOLOGY LETTERS, vol. 284, no. 1, 6 May 2008 (2008-05-06), pages 28 - 34 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170022573A1 (en) * 2014-04-11 2017-01-26 The Trustees Of The University Of Pennsylvania Compositions and Methods for Metagenome Biomarker Detection
CN106414775A (zh) * 2014-04-11 2017-02-15 宾夕法尼亚大学董事会 用于宏基因组生物标志检测的组合物和方法
US10883145B2 (en) * 2014-04-11 2021-01-05 The Trustees Of The University Of Pennsylvania Compositions and methods for metagenome biomarker detection
CN111455021A (zh) * 2019-01-18 2020-07-28 广州微远基因科技有限公司 去除宏基因组中宿主dna的方法及试剂盒
CN111455021B (zh) * 2019-01-18 2024-06-04 广州微远医疗器械有限公司 去除宏基因组中宿主dna的方法及试剂盒
EP4299713A4 (fr) * 2021-02-26 2025-01-22 Yokogawa Electric Corporation Procédé de mesure et système de mesure

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