WO2009069843A1

WO2009069843A1 - Methods for fabrication of metagenomes microarrays and quantitative detection of genes or microorganisms using metagenomes microarrays

Info

Publication number: WO2009069843A1
Application number: PCT/KR2007/006645
Authority: WO
Inventors: Jin-Woo Bae; Young-Do Nam; Ho-Won Chang; Kyoung-Ho Kim; Seong Woon Roh
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2007-11-30
Filing date: 2007-12-18
Publication date: 2009-06-04
Anticipated expiration: 2010-05-30

Abstract

The present invention relates to a method for quantitative detection of genes or microorganisms using a microarray prepared by arranging massive amount of metagenomes on a glass slide at high density, more precisely a method for quantitative detection of genes or microorganisms using a microarray on which metagenomes, the total DNA obtained from environmental samples were printed at high density after purification, fragmentation and partialization. According to the method of the present invention, tens of thousands - hundreds of thousands of samples can be analyzed quantitatively at a time without any amplification process by using a proper probe. Despite the excellent specificity, the conventional detection method is limited in detection, because the conventional method can analyze only one sample at a time, which means in order to analyze numbers of samples, analyze has to be repeated numbers of times. The method of the present invention overcomes the problem of the conventional method, so that it facilitates quantitative detection of genes (or gene groups) or microorganisms (or microorganism groups) included in tens of thousands-hundreds of thousands of samples at a time with accuracy.

Description

[DESCRIPTION] [invention Title]

METHODS FOR FABRICATION OF METAGENOMES MICROARRAYS AND QUANTITATIVE DETECTION OF GENES OR MICROORGANISMS USING METAGENOMES MICROARRAYS

[Technical Field]

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

PCR.

[Background Art] It is not only very difficult to separate viable but non-culturable (VBNC) microorganisms as pure culture but also sometimes impossible (enrichment bias) to culture themselves because of difference of substrate requirements for environmental microorganisms. Therefore, the method for detecting diversity of microorganisms based on the nucleotide sequence of 16S rRNA, the SSU rRNA (small subunit ribosomal RNA) gene of a microorganism, has been proposed without culture, and used as an index for phylogeny and taxonomy. According to the advancement of 16S rRNA gene analysis techniques based on PCR and sequencing, it has been confirmed that large numbers of microorganisms having important activities are uncultivated

(Amann et al. Microbiol Rev. 59 (1) : 143-69. 1995), and the number of those microorganism separated by human so far is just less than 1% of the total microorganisms in the nature

(Pace, Science. 276 (5313) : 734-40. 1997) .

Phylogenetic systematics based on the comparison of sequences of 16S rRNA genes plays a key role in highlighting the diversity of microorganisms, better than the conventional classification systems (Woese et al . Proc Natl Acad Sci USA. 87 (12) : 4576-4579. 1990) . Owing to phylogenetic systematics, we understood that specific microorganism groups were out there without culturing. Besides, with the advancement of ecological analysis techniques at molecular level such as DGGE (Denaturing Gradient Gel Electrophoresis) , TGGE (Temperature Gradient Gel Electrophoresis) , cloning, etc, diversity of microorganisms was acknowledged and unidentified microorganisms began to be identified. Recently, according to the introduction of high technology such as microarray and shot-gun sequencing, more environmental samples have been analyzed in a short period, contributing to broadening the knowledge of diverse microorganisms everywhere in the world. It is common agreement in reports in the field of microbiology that DNA microarray can be applied without limitation. Moreover, application of DNA microarray keeps overcoming its limit and turning directions to other fields, so that it is now applied to microorganism detection with high efficiency (Bodrossy et al., Curr OpIn microbiol, 7, 245-254, 2004) . 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. Even though 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) . To realize the general application of DNA microarray in analysis of various microorganisms, the above limits have to be overcome. Among microarrays being used these days, 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. Therefore, a novel detection method enabling precise detection to the lower level than species with high resolution is required. To respond this request, the present inventors developed a novel DNA microarray, which is GPM (genome-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).

The present inventors further confirmed that the 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) . 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. So, according to the recent studies, a method using microarray probe directly obtained from the nature by digital multiple-amplification of uncultivated microorganism genomes without culturing microorganism has been developed (Korean Patent No. 10-0759390) . However, even with the best microarray probe for the detection of genes and microorganisms obtained so far, quantitative detection of those genes or microorganisms is still very- difficult because every target gene or microorganism to be searched would be labeled and performed hybridization processes individually.

Thus, 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.

[Disclosure] [Technical Problem] It is an object of the present invention to provide a microarray including a glass board on which metagenomes comprising genomes of uncultivated microorganisms are arranged at high density, a kit for analysis comprising the microarray, a method for producing the metagenome microarray, and a method for quantitative detection of genes or microorganisms in metagenomes using the microarray.

[Technical Solution] To achieve the above object, 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:

1) extracting metagenomes from environment;

2) purifying the metagenomes extracted in step 1) ; and

3) 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;

2) hybridizing the labeled target sample of step 1) with the microarray of claim 1 and washing thereof; and

3) detecting signals from the labeled target sample from the hybridized microarray of step 2) .

In addition, the present invention provides a kit for quantitative detection of specific genes or microorganisms included in metagenomes comprising the microarray.

[Advantageous Effect]

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.

[Description of Drawings] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

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; and

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.

[Best Mode] Hereinafter, the terms used in this description are def ined .

In this invention, "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.

In this invention, "microarray" indicates the first or secondary structural array having separated sections divided regularly on the solid support. In general, 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. In this invention, "printing" indicates the fixation of metagenomes extracted from the natural environment on the microarray, which is also called "spotting".

In this invention, "labeling" indicates labeling for detecting the hybridization of a target DNA with the microarray probe.

In this invention, "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. Hereinafter, the present invention is described in detail .

The present invention also provides a microarray in which metagenomes extracted from environment or their fragments are printed on the solid support.

1) extracting metagenomes from environment;

2) purifying the metagenomes extracted in step 1); and

3) printing the metagenomes purified in step 2) on the solid support.

In this method, 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. In this method, 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. For the partialization of metagenome, SSH

(suppression subtractive hybridization) is preferably used but not always limited thereto. 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) . In a preferred embodiment of the present invention, 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. At this time, the strain for comparison is called Mriver' and the strain comprising specific DNA which dose not belong to driver is called ^λtester' . 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.

For fragmentation of metagenome, the conventional fosmid library or BAC library is preferably used but not always limited thereto. To obtain metagenome fragments, 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. As a vector in this invention, 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. In particular, BAC (Bacterial Artificial Chromosome) and 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. 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.

In this method, 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. In this invention, 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. 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:

1) labeling DNA separated from the samples;

2) hybridizing the labeled target sample of step 1) with the microarray of claim 1 and washing thereof/ and

3) detecting a signal from the labeled target sample from the hybridized microarray of step 2) .

In this method for quantitative detection, 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.

In this method for quantitative detection, 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. In the meantime, the signal amplification effect by biotin-tyrimide-Cy5 is 5 - 10 times the amplification effect of the conventional Cy5, and 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. On the contrary, according to the method using photobiotin-tyrimide-Cy5, DNA amplification process is omitted, suggesting that exact DNA hybridization can be predicted. After spotting the metagenomes extracted from human fecal samples on a microarray, the present inventors performed labeling of the target DNA, followed by hybridization. As a result, the strong signal was observed on the metagenome on the slide that corresponds to the labeled metagenome in real array (see Figs. 2 and 3) . The present inventors obtained nucleotide sequences of total 16S ribosomal gene of bacteria, E. coli group,

Firmicutes group, and Bacteroides group from the NCBI

(National Center for Biotechnology Information) nucleotide sequence database and then constructed sequence probe for each group (see Table 2) . The present inventors confirmed that when a specific microorganism or a microorganism group sequence was hybridized with the metagenome microarray as a probe, the amount of the specific microorganism can be calculated quantitatively on the metagenome microarray (see

Figs 5 and 6) .

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) .

In this method, 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. In genera], formamide or dimethyl sulfoxide is used as a denaturant included in hybridization buffer. The higher the concentration of a denaturant is, the higher the sensitivity of hybridization is. As for the concentration of such denaturant, formamide is preferably included by 10- 70% and more preferably included by 30-50 %, but not always limited thereto. Considering the sensitivity of hybridization, temperature for hybridization and concentration of salts in washing solution can be regulated. In general, 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.

In this method, the detection of step 3) can be differently executed according to the method of DNA labeling. But preferably, 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. As a detection system, CCD or confocal laser in light launching part can be used. Nucleic acid derived from the sample can be labeled by Cy3 or Cy5. At this time, 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. Besides, 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. However, 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. However, 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.

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

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.

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

International, Inc., Sunnyvale, CA) . In the case that a sample had higher concentration than that, it was diluted with 0.1 x TE buffer. The metagenome was distributed to a

384-well microplate (5 ul/well) , to which the same amount of 2x microarray spotting solution (ArrayltTM, Telechem International, Inc., Sunnyvale, CA) was added and mixed well, followed by printing. As shown in Fig. 1, the metagenome was printed on a slide in a consistent order, 4 sets (total 216 metagenomes) for reliable statistic results. The precise location of metagenome DNA printed on the slide is shown in Table 1. For stronger DNA binding, the microarray was cross-linked by irradiating 120 mJ of UV. Then, the slide was reacted with 0.17 M succinic anhydride dissolved in the mixed solution comprising 240 ml of 1- methyl-2-pyrrolidinone and 10.7 ml of 1 M boric acid solution to inactivate remaining residues. Right after inactivation, DNA was denatured by boiling for 2 minutes, which was sunk in cold 95% ethanol, air-dried at room temperature and stored in dark place.

[Table l]

Spot location and sample name of the human fecal metagenome microarray

Example 2 : Labeling of metagenome and microorganism group specific nucleotide (sequence probe) and hybridization

The present inventors performed labeling as follows.

First, 1 ug of metagenome was labeled by reacting with 2.5 mM Cy5 dCTP (Amersham Pharmacia Biotech, Piscataway, NJ) using BioPRime DNA labeling system at 37 ^°C for 3 hours. The labeled target DNA was purified by QIAquick PCR purification column (Qiagen, Valencia, CA) . Then, the DNA was completely dried and concentrated by SpeedVac. The DNA was resuspended in 4.35 ul of distilled water and used for hybridization.

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) .

10 ul (1 ug) of microorganism group specific nucleotide

(sequence probe) was treated wibh 3 ul (1 mg/ml) of photobiotin. The photobiotin was activated by irradiating 120 mJ of UV for conjugation to the nucleotide. Non- conjugated photobiotin was eliminated by ethanol precipitation. The photobiotin-nucleotide purified by ethanol precipitation was dissolved in 10 ul of distilled water and 4.35 ul (435ng) of labeled nucleotide was used for the reaction. Every hybridization of microarray was performed triplicate for statistic analysis. After hybridization, each microarray slide was washed with solution 1 (ISSC and 0.2% SDS) and cover slip was removed. The slide was washed stepwise with washing solution 1 , washing solution 2 (O.lx SSC and 0.2% SDS), and washing solution 3 (O.lx SSC), 5 minutes each, and dried by using a centrifuge.

Microorganism specific nucleotide (sequence probe) 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. 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.Oβx SSC solution. The slide was dried by using a centrifuge .

[Table 2]

Nucleotide sequence of group specific probe for microorganism identification

Example 3: Microarray scanning and data analysis

For scanning of microarray finished with the reaction, GenePix 4000B microarray scanner set (Axon instruments, Union City, CA) was used. For consistent scanning of all hybridized slides, gains of laser power and photomultiplier tube (PMT) were 100%. Scanned images were stored as 16 bit TIFF files. Pixel density (intensity) of each hybridized spot was quantified by GenePix software 4.1 (Axon instruments) . The optical expression of the result of hybridization was controlled automatically by the software containing the standardized images. Each lattice of circle indicating exact location of each DNA spot on array was quantified and changed into fluorescence spot image. The signal intensity for each spot was automatically determined. Local background signal was subtracted from hybridization signal on each spot. SNR (signal-to-noise ratio) values were calculated by the following formula.

SNR = Signal Intensity - Background Intensity / Standard Deviation of Background

Signal intensity: intensity of light of each spot Background intensity: intensity of light of background Standard deviation of background: standard deviation of background intensity

"Background intensity" was investigated by measuring the intensity of local spot. "Standard deviation of background" was calculated with every pixel using GenePix software. Statistic analysis was performed by using Excel 2003 (Microsoft) and Sigmaplot 8.0 (Jandel Scientific, San Rafael, CA) . As a result, when metagenomes extracted from environment were directly spotted on a microarray and the target metagenome DNA was labeled and hybridized with the microarray, as shown in Fig. 2, those metagenomes on the slide corresponding the metagenomes labeled on real array exhibited strong signals. In the case of Al, when its metagenome (target sample) was labeled on the microarray, it exhibited 100% signal of its own. 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. In the case of Gl and Hl, their comparative groups (G2 - G6, H2 - Hβ) exhibited high signals, compared with other samples (at least 82% except H5, and at least 78% except G2) . That was because those samples were taken from younger samples that suggested they were less affected by outside factors so that metagenomes were reserved similarly. Therefore, the method of the invention presents the relativity of microorganisms quantitatively, as a whole, which the conventional method for analyzing microorganisms by PCR could not (Figs. 2 and 3) .

According to the present invention, quantitative detection of a specific microorganism was possible on the metagenome microarray by hybridizing a specific microorganism or a specific microorganism group specific sequence probe with the metagenome microarray. In the case of A5, B5, C5, D5, E5, F5, G5 and H5, sequence probe targeting the entire bacteria was used and the calculated value was considered as 100%. Then, the amounts of Bacteroides, Firmicutes, and E. coli included in samples were calculated by one time experiment (Figs. 5 and 6) .

It was also confirmed that when photobiotin-TSA-Cy5 was used, instead of the conventional Cy5 dUTP labeling, signal of probe hybridized on the microarray was significantly increased (Fig. 7) . The present inventors, therefore, established a method for detecting interrelation among metagenomes by the metagenome microarray and further confirmed that the method enables quantitative detection of a specific microorganism included in the metagenome microarray by using a specific probe. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS]

[Claim l]

A microarray in which metagenomes extracted from environment or their fragments are printed on the solid support.

[Claim 2]

The microarray according to claim 1, wherein the metagenome is obtained from one or more places selected from the group consisting of water, sea, soil, air, foods, waste water and intestines or tissues of animal including human and plants.

[Claim 3] The microarray according to claim 1, wherein the solid support is selected from the group consisting of slide glass, silicon chip and nitrocellulose or nylon membrane .

[Claim 4]

The microarray according to claim 1, wherein the printing is performed by the method selected from the group consisting of microspotting using a pin, microdropping based on inkjet principle and addressing using electricity. [Claim 5]

A kit for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the microarray of claim 1.

[Claim β]

A method for producing a microarray for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the following steps: 1) extracting metagenomes from environment;

2) purifying the metagenomes extracted in step 1); and

3) printing the metagenomes purified in step 2) on the solid support.

[Claim 7]

The method for producing a microarray according to claim 6, wherein the metagenome of step 1) is obtained from one or more places selected from the group consisting of water, sea, soil, air, foods, waste water and intestines or tissues of animal including human and plants.

[Claim 8]

The method for producing a microarray according to claim 6, wherein the purification of step 2) is performed by ethanol precipitation or using DNA separation kit.

[Claim 9]

The method for producing a microarray according to claim 6, wherein the metagenome purified in step 3) is additionally partialized or fragmented.

[Claim 10]

The method for producing a microarray according to claim 9, wherein the partialization is performed by SSH

(Suppression Subtractive Hybridization) and fragmentation is performed by using BAC library (bacterial artificial chromosome library) or fosmid library.

[Claim 11]

The method for producing a microarray according to claim 6, wherein the solid support of step 3) is selected from the group consisting of slide glass, silicon chip and nitrocellulose or nylon membrane.

[Claim 12]

The method for producing a microarray according to claim 6, wherein the printing of step 3) is performed by the method selected from the group consisting of microspotting using a pin, microdropping based on inkjet principle and addressing using electricity.

[Claim 13]

A method for quantitative detection of specific genes or microorganisms included in metagenomes, comprising the following steps:

1) labeling DNA separated from the samples;

2) hybridizing the labeled target sample of step 1) with the microarray of claim 1 and washing thereof; and 3) detecting a signal from the labeled target sample from the hybridized microarray of step 2) .

[Claim 14]

The method for quantitative detection according to claim 13, wherein the sample of step 1) is obtained from one or more sources selected from the group consisting of environment, human intestines, animal intestines, water and foods .

[Claim 15]

The method for quantitative detection according to claim 13, wherein the labeling of step 1) is performed 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 .

[Claim 1 6]

The method for quantitative detection according to claim 15, wherein the labeling is performed by using photobiotin-tyrimide-Cy5.