CN103060309B - Extraction method for metagenome - Google Patents
Extraction method for metagenome Download PDFInfo
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Abstract
The present invention discloses an extraction method for metagenome, and a method of probe hybridization-bead capture is used to reduce the host DNA content in a host metagenomic DNA sample. According to the method, the Alu repeat sequence of the host DNA sequences and conserved sequence at both ends thereof are used as templates to design one-way or two-way probes, wherein the 5 'end of each probe is modified with biotin, streptavidin coated beads are used to capture host DNA hybridized with probes, in order to achieve aims of weakening host DNA background in a metagenome, and enhancing sequencing data efficiency.
Description
Technical field
The present invention relates to and extract grand genomic method, relate to the method reducing host DNA in host's macro genome DNA sample particularly.
Background technology
Grand genome is all genetic material appeared in a certain environmental sample, comprises the genome of many individual species.Metagenomics refers to and directly from environmental sample, obtains genetic material, to the science that grand genome is studied.Along with the development of high-throughput DNA sequencing and information biology, the using value of metagenomics constantly promotes.By analyzing grand genomic library, population characteristic and the interaction rule of microorganism in certain environment can be found, inquiring into influencing each other of microorganism and environment.To the order-checking of microbial population DNA, the gene with specific function can be screened, obtain the albumen of specific function.
Human respiratory touches the microorganisms such as a large amount of virus and bacterium every day, effectively can understand the feature of each microbial population to the grand genomic research of respiratory tract, thus helps clinical diagnosis and treatment.In the grand genome of respiratory tract, relative to the genome content of host people, pathogen gene group content is generally smaller.Therefore, in the grand gene order-checking of respiratory tract, for reaching the enough sequence coverage of pathogen gene group, the impact eliminating host genome is very important.
In human respiratory sample, the quality of STb gene is the key of grand gene order-checking success or not.To transmissible disease sample, the method for process DNA pollution needs to spend many time, or needs special treatment process when collecting sample.During general extraction DNA, need to utilize washing agent SDS or the lysing cell such as N,O-Diacetylmuramidase, Proteinase K, then utilize phenol/chloroform, last alcohol settling DNA.When extracting macro genome DNA, due to cell type diversity and there is the interference of host DNA, so need more effective purification process for the extraction of DNA.
People's Alu repeated sequence belongs to short and is dispersed in sequence (short interspersed elements, SINEs) family.The length of each Alu sequence is about 280bp, has variable poly A(poly-A) tail.Alu repeated sequence, more than 1,000,000, accounts for 10% of human genome, has been used to the analysis of some genetic diseases of the mankind.Alu repeated sequence is different, can be divided into several subfamily, is fabulous mark, can be used to detect human genome.Alu, as special probe, uses round pcr, can quantitative human gene group DNA.In human genome, many new Alu sequences are found, and most of sequence does not exist in other primates genomes, and the analysis therefore according to special Alu repeated sequence can be used as human DNA qualification and quantitative favorable method.
The magnetic bead of Streptavidin bag quilt, has been widely used in biotinylation nucleic acid, antibody or other biological elementization part at present and target molecule is separated and process.Diameter is the super-paramagnetic bead of 2.8 μm, has to be covalently attached to surface and in addition with the individual layer restructuring Streptavidin that BSA closes.Owing to having the Streptavidin of individual layer, most biotin-binding site spatially not only can in conjunction with free biotin, but also can in conjunction with biotinylated part.They demonstrate fast performance liquid reacting dynamics characteristic, and the specific surfaces that shape is determined is convenient to carry out efficient capture, separation and downstream process.
Real-time fluorescence quantitative PCR (Real-time polymerase chain reaction, be called for short Real-time PCR, be also referred to as quantitative Real-time polymerase chain reaction, be called for short Q-PCR), based on polymerase chain reaction (polymerase chain reaction, be called for short PCR) Protocols in Molecular Biology, this technology carries out quantitative analysis to target dna molecule while can increasing to target fragment.Real-time fluorescence quantitative PCR uses special probe sequence or primer, along with the increase of DNA profiling amount in amplification procedure, the fluorescence intensity discharged is also in increase, and therefore real-time fluorescence quantitative PCR can utilize less sample, the content of detection of dynamic initial target DNA.
Summary of the invention
The object of the invention is that providing a kind of extracts the grand genomic method of the mankind, and the method utilizes the probe hybridization method of residues, effectively can reduce host DNA background interference in the grand genome of host.
First aspect present invention, provides a kind of grand Extraction Methods of Genome, it is characterized in that utilizing probe hybridization magnetic capture mode to reduce the impact of host DNA in host's macro genome DNA sample.
According to the embodiment of the present invention, described probe and described host genome DNA sample hybridize rear and described magnetic bead coupling.
According to the embodiment of the present invention, provide a kind of grand Extraction Methods of Genome, comprise step:
1) described host's macro genome DNA sample of the described probe and extraction that are marked with vitamin H is hybridized;
2) with the complex body of the described magnetic capture probe and described host DNA that are coated with Streptavidin, and magnetic bead-probe-target DNA mixture is separated with magnetic frame;
3) the magnetic bead scavenging solution after the hybridization solution of removal described magnetic bead-probe-target DNA mixture and hybridization is carried out purifying and reclaim DNA.
According to the embodiment of the present invention, after described probe and described magnetic bead coupling with the hybridization of described host genome.
According to another implementation of the invention, provide a kind of grand Extraction Methods of Genome, comprise step:
1) the described probe conjugate of described magnetic bead and the mark vitamin H being coated with Streptavidin is utilized;
2) by mixture good for coupling and described host's macro genome DNA sample hybridization;
3) remove magnetic bead-probe-target DNA mixture, purifying is carried out to hybridization solution and hybridization scavenging solution and reclaims DNA.
According to the embodiment of the present invention, in described grand Extraction Methods of Genome, at coupling temperature, use multiple forward and reverse probe.Use multiple forward and reverse probe, the scope that probe is combined when host DNA is hybridized can be expanded, simultaneously can Streptavidin site on abundant saturated magnetic bead, improve the utilising efficiency of magnetic bead.
According to the embodiment of the present invention, in described grand Extraction Methods of Genome, after hybridization is complete, described magnetic bead-probe-DNA mixture is heated or sodium hydroxide method sex change, reclaim the magnetic bead that coupling has probe.Reclaim the magnetic bead that coupling has probe, can experimental cost be reduced.
According to the embodiment of the present invention, for human genome Alu repeated sequence family designing probe, cut down by these probe hybridizations, reach the object reducing human DNA ratio in the sample to which.Its middle probe is with the conserved sequence at multiple Alu repeated sequence family and its two ends for template, and bi-directional design probe, probe length is between 40 ~ 70nt, and 5 ' terminal modifiedly has vitamin H.The optional people's Alu sequence from sequence table SEQ ID NO:1 ~ 139 of probe sequence.Utilize the Alu sequence that people is special, eliminate the impact of host's human genome in respiratory tract sample, the grand gene order-checking of high-throughput is had to the help of highly significant.
Fourth aspect present invention provides respiratory tract sample treatment, for different sampling method, sample physical properties, adopts the methods such as dilution liquefaction, collected by centrifugation, the cell in maximum efficiency enriched sample.
The method is not only applicable to study for human respiratory metagenomics, also other positions of human body such as oral cavity, digestive tube, skin, the research of reproductive tract metagenomics is applicable to, effectively can reduce the high background interference of people DNA, thus effective enriched microorganism DNA, reduce the sequencing data waste that host DNA background causes, improve the quantum of output of valid data, microbial information in the grand genome of more deep excavation.
Accompanying drawing explanation
Fig. 1 is segmental bronchus and bronchoalveolar lavage fluid and airway secretions STb gene gel electrophoresis figure.
Fig. 2 is the qPCR typical curve that plasmid standard is drawn.
Fig. 3 is plasmid qPCR absolute quantitation amplification curve in biased sample stoste in embodiment 1, hybridization solution Ethylene recov and magnetic bead scavenging solution.3 groups of set of curves distinguish representative simulation hybrid dna former state from left to right, and hybridization solution BW, scavenging solution W, horizontal line represents threshold line.
Embodiment
experimental example bronchoalveolar lavage fluid (BAL), airway secretions and whole blood total DNA extraction
One, bronchoalveolar lavage
Select middle lobe of right lung (B4 or B5) or left lung tongue section to bronchoalveolar lavage fluid cell, limitation tuberculosis becomes and then carries out lavation in homologous bronchi lung section.First want the lung section of lavation to inject 2% lignocaine 1 ~ 2ml through biopsy hole by thin silicone tube, do the toponarcosis of lavation lung section; Then by the Bronchofiberscope top closely section of wedging or sub-segmental bronchus opening part, then 37 DEG C of sterile salines are injected through biopsy hole fast by silicone tube.Each 25 ~ 50ml, total amount 100 ~ 250ml, is generally no more than 300ml; Use 50 ~ 100mmHg vacuum suction to reclaim irrigating solution immediately, the usual rate of recovery is 40% ~ 60%; By recovering liquid immediately by double-layer sterile filtered through gauze removing mucus, and record total amount; Load silicon plastic bottle or be coated with in silicon sterilizing Glass Containers (minimizing cell adhesion), being placed in the vacuum flask containing ice cube.
Two, airway secretions attracts
Select suitable sputum aspirator tube, the external diameter of sputum aspirator tube should not exceed tracheal catheter internal diameter 1/2; Check that whether sputum aspirator is intact, attract negative pressure to be no more than-50mmHg; The swollen lung of pure oxygen is carried out, tracheal lavage after cleaning oral cavity.Block sputum aspirator tube negative pressure, sputum aspirator tube is inserted tracheal catheter and do, exhaust after suction sputum pars oralis pharyngis secretory product., put forward the open negative pressure of 0.5cm on when reaching tracheal catheter end, rotation carried; Suction sputum action is soft, rapidly, each suction sputum time is no more than 15 seconds; Strict aseptic technique.
Three, instrument and reagent prepare
ABI 7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems, Life Technologies company).
Qubit
2.0 fluorescent quantitation instrument (Invitrogen, Life Technologies company).
Proteinase K: 100 mg Proteinase Ks are dissolved in 5 mL distilled waters, are mixed with the solution of 20 mg/ml ,-20 DEG C of preservations.
1L PBS damping fluid (pH7.4):
Potassium primary phosphate (KH
2pO
4) (purchased from Beijing Chemical Plant), 0.27g
Sodium phosphate dibasic (Na
2hPO
4) (purchased from western Gansu Province chemical industry), 1.42g
Sodium-chlor (NaCl) (purchased from western Gansu Province chemical industry), 8g
Repone K (KCl) (purchased from Beijing Chemical Plant), 0.2g
Add the abundant stirring and dissolving of deionized water about 800 mL, then add concentrated hydrochloric acid adjust pH to 7.4, last constant volume to 1L, room temperature preservation after autoclave sterilization.
DNA extraction kit: QIAamp DNA Mini Kit(QIAGEN, article No. 51306).
Four, extraction step
1, get irrigating solution and collect 1 ~ 5ml, 13000rpm centrifugal concentrating 10 minutes, abandon supernatant, add equal-volume PBS and dilute, assist in dissolving is disperseed, and for the airway secretions of thickness, needs first to use 2 ~ 3 times of volume PBS to dilute, and homogeneity is carried out in soft mixing;
2, prepare some 1.5ml centrifuge tubes, often pipe adds 50 μ l Proteinase Ks in pipe;
3, often pipe add 500 μ l dilute dispersion after respiratory secretions sample, supply less than 500 μ l PBS;
4, often pipe adds 500 μ l buffer AL(QIAamp DNA Mini Kit), soft mixing is placed in 56 DEG C of water-baths for 15 seconds, hatches 10 minutes;
5, after taking out, centrifugal fast, drop tube wall and pipe covered is collected in return pipe, add 500 μ l dehydrated alcohols, vortex mixes 15 seconds, after centrifugal fast, be transferred in centrifugal column by entire mixture in pipe and (can carry out at twice), centrifugal 1 minute of 8000rpm, in transfer centrifugal column to new centrifuge tube;
6, add 500 μ l buffer AW1, centrifugal 1 minute of 8000rpm, discards liquid in pipe;
7, add 500 μ l buffer AW2, centrifugal 3 minutes of 14000rpm, after discarding liquid in pipe, sky gets rid of 1 minute;
8, shift centrifugal column in the new centrifuge tube of 1.5ml, add 30 μ l distilled waters, after room temperature places 2 minutes, centrifugal one minute of 8000rpm.
The STb gene extracted, through agarose gel electrophoresis analysis, the results are shown in Figure 1, M:DL2000, and band is descending is 2000bp, 100bp, 750bp, 500bp, 250bp, 100bp.The DNA that swimming lane 1 and swimming lane 2 obtain for airway secretions extraction, concentration is respectively 72ng/ μ l, 15ng/ μ l; The DNA that swimming lane 3 and swimming lane 4 obtain for bronchoalveolar lavage fluid extraction, concentration is 21ng/ μ l.As can be seen from Figure 1: genomic dna band is complete.
Fig. 2 is the qPCR typical curve that plasmid standard is drawn.Standard substance adopt BAP plasmid (2.6 kbp) 5 times of gradient dilutions to become the sample of 5 concentration gradients, by standard concentration and Ct value drawing standard curve, for plasmid content in quantitative analysis sample.In figure, X-coordinate Log CO represents template concentrations, and ordinate zou Ct represents the Ct value of sample, and each square dot represents standard model.To carry out in simulated experiment before and after hybridization the quantitative of plasmid in sample thus.
Irrigating solution and whole blood carry out total DNA extraction with reference to above-mentioned steps.
embodiment hybridization abatement reduces host DNA background in macro genome DNA
The present invention utilizes the Alu sequence feature that the frequency of occurrences is high in people DNA, for Alu family sequence and two ends conservative region designing probe, 5 ' end biotin labeling, and by not homotactic multiple probe with the use of, by the magnetic bead of avidin bag quilt together, hybridization compound DNA is caught out, thus reduces the background interference of host DNA in grand genome.
Required reagent and mother liquor:
20 × SSPE: take 210.6g NaCl(purchased from western Gansu Province chemical industry), 27.6g NaH
2pO
4 .h
2o(is purchased from Beijing Chemical Plant), 5.845g EDTA(is purchased from western Gansu Province chemical industry), deionized water dissolving, adjustment pH to 7.7, is settled to 1L, filtration sterilization packing, 4 DEG C of preservations.
100 × Denhardt ' s regent solution: take 2g ficoll (Ficoll, 400 types, purchased from Sigma), 2g polyvinylpyrrolidone (PVP-40, purchased from Sigma, P-3004), 2g BSA(is purchased from rope Bora biotechnology), adding water to cumulative volume is 100ml.Filtration sterilization, is distributed into aliquot ,-20 DEG C of storages.
20 × SSC: take 88.2g Trisodium Citrate (purchased from western Gansu Province chemical industry), 175.3g NaCl, adds 900ml water dissolution, with NaOH(purchased from Beijing Chemical Plant) regulate pH to 7.0, deionized water is settled to 1L, filtration sterilization.10%SDS solution: get 10gSDS(completely newly open up purchased from Beijing reach science and technology), deionized water dissolving, is settled to 100ml.
1M Tris-HCl; Take 72.66gTris-base(completely newly open up purchased from Beijing reach science and technology), add 500ml deionized water, with HCl(purchased from Beijing Chemical Plant) regulate pH to 7.5, be settled to 600ml.
50mM EDTA solution: take 1.86gEDTA and be dissolved in 100ml deionized water.
4M NaCl solution: take 23.376gNaOH and be dissolved in 100ml deionized water.
Above mother liquor all uses filtration method degerming.
Hybridization solution: 50ml 2 × B & W buffer: get 0.5ml 1M Tris-HCl (pH 7.5), 1ml 50mM EDTA, 25ml 4M NaCl, adds water to 50ml.
50ml 2 × hybridization solution HYB buffer: get 25ml 20 × SSPE solution, 5ml 100 × Denhardt ' s solution, 10ml 50mM EDTA, 1ml 10% SDS, adds water to 50ml.
50ml magnetic bead scavenging solution wash buffer: get 2.5ml 20 × SSC, 0.5ml 10%SDS, adds water to 50ml
Probe sex change magnetic bead Ethylene recov (0.15M NaOH): take 0.6gNaOH, is dissolved in 100ml deionized water, and packing is preserved.
embodiment 1 carries out probe hybridization abatement (first coupling after hybridization) to simulation biased sample
1, simulate hybrid dna to get 20 μ l(and mix according to Nanogram Amounts 9: 1 with 2815bp plasmid by people's whole blood DNA, total amount is about 100ng/ μ l) in 200 μ l pipes, be placed in PCR instrument 95 DEG C of preheatings 5 minutes, 65 DEG C 5 minutes; Get 27 μ l 2 × hybridization solution HYB and be placed in PCR instrument 65 DEG C of preheatings 5 minutes;
2, each 0.1 μ l, the about 70pmol of probe that sequence number is SEQ ID NO:1 ~ 70 is got, 65 DEG C of preheatings 2 minutes;
3, mix above-mentioned system totally 54 μ l, 65 DEG C, hybridize 1 hour;
4, magnetic bead M-280(invitrogen is got) 30 μ l, clean 2 times with 2 × B & W buffer, abandon supernatant, resuspended with 54 μ l 2 × B & W;
5, add in magnetic bead by hybridization system (54 μ l), mixing, under room temperature, (20 DEG C ~ 25 DEG C) hatch 15 minutes, and period flicks tube wall, mixing;
6, after magnetic bead cohesive process terminates, be placed on magnetic frame by 1.5ml centrifuge tube, room temperature is placed 1 ~ 2 minute, after treating that magnetic bead is adsorbed onto tube wall, draws supernatant and retains, be labeled as BW;
7, with 100 μ l wash buffer, piping and druming cleaning magnetic bead, is then placed on magnetic frame static gently, and supernatant sucking-off reservation is labeled as W, then repeats 2 times;
8, with 100 μ l wash buffer, piping and druming cleaning magnetic bead, is then placed on magnetic frame static gently, and supernatant sucking-off reservation is labeled as W, then repeats 2 times; Ethylene recov W Qiagen PCR purification kit (QIAquick PCR Purification Kit, QIAGEN, article No. 28106) post after being cleaned by the hybridization solution BW returned and wash buffer reclaims DNA, 50 μ L eluted dnas.Magnetic bead can be preserved by following method: the resuspended magnetic bead of 20 μ l 0.15M NaOH solution, and incubated at room temperature 10 minutes, is placed on magnetic frame and removes supernatant after 1 ~ 2 minute, adds 30 μ l 1 × B & W buffer solution, preserves magnetic bead for 4 DEG C.Magnetic bead, with after 50 μ L 1 × SSC solution cleanings, adds the resuspended magnetic bead of 50 μ L 1 × SSC, hatches 5 minutes for 95 DEG C, is placed on magnetic frame and leaves standstill after 1 ~ 2 minute, remove supernatant, adds 30 μ L 1 × B & W solution and preserves.
embodiment 2 pairs of respiratory tract irrigating solution sample total DNA carry out probe hybridization abatement (first coupling after hybridization)
Except in embodiment 1 step 1, select human respiratory irrigating solution sample DNA, get in 2 × hybridization solution HYB 32 μ l, step 2 that to get sequence number be that each 3 μ l(of probe of SEQ ID NO:1 ~ 4 are about 120pmol), resuspended with 64 μ l 2 × B & W in step 4 beyond, first hybridize rear conjugated probes hybridization abatement in the same manner as in the example 1 with condition.
embodiment 3 carries out probe hybridization abatement (hybridizing after first coupling) to simulation biased sample
1, magnetic bead M-280(invitrogen is got) 30 μ l, clean 2 times with 2 × B & W buffer, abandon supernatant, add the resuspended magnetic bead of 15 μ l 2 × B & W buffer;
2, in resuspended magnetic bead, add sequence number is that each 0.1 μ l(of probe of SEQ ID NO:71 ~ 139 is about 70pmol) and 8 μ l ddH
2o, incubated at room temperature 15 minutes, period gently revolves mixing; Be placed in magnetic frame room temperature to place 1 ~ 2 minute, remove supernatant;
3, simulate hybrid dna to get 20 μ l(and mix according to Nanogram Amounts 9: 1 with about 2.6kbp plasmid by people's whole blood DNA, total amount is about 100ng/ μ l) in 200 μ lPCR pipes, be placed in PCR instrument 95 DEG C of preheatings 5 minutes, 65 DEG C 5 minutes; Get 26 μ l 2 × hybridization solution HYB and be placed in PCR instrument 65 DEG C of preheatings 5 minutes;
4, add in magnetic bead by hybrid dna and hybridization solution, mixing, is placed in PCR instrument 65 DEG C, hybridizes 1 hour, period interval mixing;
5, hybridization terminate after step with reference to the step 6-8 of embodiment 1.
embodiment 4 pairs of respiratory tract irrigating solution sample total DNA carry out probe hybridization abatement (hybridizing after first coupling)
Except adding each 1 μ l(120pmol of probe that 12 kinds of sequence numbers are SEQ ID NO:1 ~ 12 in step 2) and 3 μ l water, get except human respiratory irrigating solution sample DNA sample 20 μ l in step 3, carry out hybridization probe hybridization abatement after first coupling with method in the same manner as in Example 3 and condition.
test example 1 measures the reduction of host DNA before and after hybridization abatement
Utilize real-time fluorescence quantitative PCR to carry out absolute quantification analysis, measure the content difference of plasmid DNA in biased sample before and after hybridization abatement, and the proportional difference of people DNA and plasmid DNA.Real-time fluorescence quantitative PCR reagent adopts THUNDERBIRD Probe qPCR Mix(Toyobo), the probe of the sequence number choosing the design of particular sequence on plasmid to be the primer of SEQ ID NO:142 and SEQ ID NO:143 and sequence number be SEQ ID NO:144,5 ' end flag F AM fluorescence group of probe, 3 ' end mark TAMRA cancellation group.Realtime-PCR experiment is carried out according to THUNDERBIRD Probe qPCR Mix specification sheets.Carry out absolute quantitation by standard to the content of plasmid DNA before and after hybridization, 3, each sample repeats sample.After the STb gene content of Q-bit fluorescent quantitative measurement DNA sample, deduct plasmid DNA amount by total amount, be namely people's DNA content, measure in units of ng, the results are shown in Table 1.
Fig. 3 is plasmid qPCR absolute quantitation amplification curve in biased sample stoste in embodiment 1, hybridization solution Ethylene recov and magnetic bead scavenging solution.3 groups of set of curves distinguish representative simulation hybrid dna former state, hybridization solution BW, scavenging solution W from left to right.Green horizontal line represents threshold line, and the cycle number that threshold line is corresponding with amplification curve intersection is Ct value, can be compared calculate initial DNA profiling amount by Ct value.Ct value is less, and initial template concentration is larger, can calculate original template total amount in conjunction with DNA solution volume.
Through real-time fluorescence quantitative PCR, absolute quantification analysis is carried out to the plasmid in hybrid dna stoste and hybridization Ethylene recov, merge the plasmid amount obtained after BW and W purifying reclaims.In embodiment 1, plasmid organic efficiency is about 91% of plasmid in initial mixing DNA, and descendant and plasmid DNA amount (ng) ratio of hybridization are about 1: 1; In embodiment 3, plasmid organic efficiency is about 83.5% of plasmid in initial mixing DNA, and descendant and plasmid DNA amount (ng) ratio of hybridization are about 0.92: 1, effectively can reduce the content of people DNA, the results are shown in Table 1.
Plasmid DNA before and after hybridizing in table 1 quantitative analysis embodiment 1 and 3 and people's DNA content and ratio.
test example 2 measures DNA of bacteria content
The conservative region implementation sequence number of the 16S rDNA of directed toward bacteria and ancient bacterium is the universal primer of SEQ ID NO:140 and SEQ ID NO:141, object is the real-time fluorescence quantitative PCR by dye method, detects the change of the relative content of microorganism in respiratory tract irrigating solution STb gene before and after hybridization.Life science portion of Realtime-PCR(TOYOBO company limited is carried out, Osaka, Japan, article No. QPS-201 according to THUNDERBIRD SYBR qPCR Mix specification sheets) experiment.Because initial DNA usage quantity is 20 μ l, reclaiming wash-out water consumption after hybridization is 40 μ l, therefore in Real-time PCR quantitative experiment, reclaims 2 times that DNA consumption volume is DNA consumption volume before hybridization, to adjust the difference of the two original bulk volume ratio after hybridization.
Through Real-time PCR quantitative experiment, in embodiment 2 and embodiment 4, the bacteria content after hybridization abatement improves about 3 times and 16 times, respectively in table 2.Use this method, can reuse coupling has the magnetic bead of probe to carry out hybridization abatement to actual respiratory tract irrigating solution sample, reduces the cost of sample hybridization process.Before and after bacteria content hybridization, ratio is in table 2.
Bacteria content before and after table 2 quantitative analysis embodiment 2 and embodiment 4 sample hybridization
* the front sample Ct value of sample Ct value-hybridization after ddCt=hybridization
* content ratio (the rear DNA of bacteria content of hybridization: DNA of bacteria content before hybridization)=2
-ddCt.
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ctactacta 69
<210> 17
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 17
ggaggaggag gaggaggagg aggaggagga ggaggaggag gaggaggagg aggaggagga 60
ggaggagga 69
<210> 18
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 18
gaagaagaag aagaagaaga agaagaagaa gaagaagaag aagaagaaga agaagaagaa 60
gaagaagaa 69
<210> 19
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 19
tcatcatcat catcatcatc atcatcatca tcatcatcat catcatcatc atcatcatca 60
tcatcatca 69
<210> 20
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 20
ccaccaccac caccaccacc accaccacca ccaccaccac caccaccacc accaccacca 60
ccaccacca 69
<210> 21
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 21
tagatagata gatagataga tagatagata gatagataga tagatagata gatagataga 60
tagatagat 69
<210> 22
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 22
caaacaaaca aacaaacaaa caaacaaaca aacaaacaaa caaacaaaca aacaaacaaa 60
caaacaaac 69
<210> 23
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 23
taaataaata aataaataaa taaataaata aataaataaa taaataaata aataaataaa 60
taaataaat 69
<210> 24
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 24
gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa 60
gaaagaaag 69
<210> 25
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 25
tccatccatc catccatcca tccatccatc catccatcca tccatccatc catccatcca 60
tccatccat 69
<210> 26
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 26
cgaacgaacg aacgaacgaa cgaacgaacg aacgaacgaa cgaacgaacg aacgaacgaa 60
cgaacgaac 69
<210> 27
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 27
tgaatgaatg aatgaatgaa tgaatgaatg aatgaatgaa tgaatgaatg aatgaatgaa 60
tgaatgaat 69
<210> 28
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 28
cggacggacg gacggacgga cggacggacg gacggacgga cggacggacg gacggacgga 60
cggacggac 69
<210> 29
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 29
ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa 60
ggaaggaag 69
<210> 30
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 30
tacatacata catacataca tacatacata catacataca tacatacata catacataca 60
tacatacat 69
<210> 31
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 31
tcaatcaatc aatcaatcaa tcaatcaatc aatcaatcaa tcaatcaatc aatcaatcaa 60
tcaatcaat 69
<210> 32
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 32
gacagacaga cagacagaca gacagacaga cagacagaca gacagacaga cagacagaca 60
gacagacag 69
<210> 33
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 33
cctacctacc tacctaccta cctacctacc tacctaccta cctacctacc tacctaccta 60
cctacctac 69
<210> 34
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 34
gcccgcccgc ccgcccgccc gcccgcccgc ccgcccgccc gcccgcccgc ccgcccgccc 60
gcccgcccg 69
<210> 35
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 35
ttaattaatt aattaattaa ttaattaatt aattaattaa ttaattaatt aattaattaa 60
ttaattaat 69
<210> 36
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 36
gggagggagg gagggaggga gggagggagg gagggaggga gggagggagg gagggaggga 60
gggagggag 69
<210> 37
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 37
ggcaggcagg caggcaggca ggcaggcagg caggcaggca ggcaggcagg caggcaggca 60
ggcaggcag 69
<210> 38
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 38
gccagccagc cagccagcca gccagccagc cagccagcca gccagccagc cagccagcca 60
gccagccag 69
<210> 39
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 39
caaaacaaaa caaaacaaaa caaaacaaaa caaaacaaaa caaaacaaaa caaaacaaaa 60
caaaacaaa 69
<210> 40
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 40
tggaatggaa tggaatggaa tggaatggaa tggaatggaa tggaatggaa tggaatggaa 60
tggaatgga 69
<210> 41
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 41
taaaataaaa taaaataaaa taaaataaaa taaaataaaa taaaataaaa taaaataaaa 60
taaaataaa 69
<210> 42
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 42
gcccagccca gcccagccca gcccagccca gcccagccca gcccagccca gcccagccca 60
gcccagccc 69
<210> 43
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 43
gctcagctca gctcagctca gctcagctca gctcagctca gctcagctca gctcagctca 60
gctcagctc 69
<210> 44
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 44
gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa gaaaagaaaa 60
gaaaagaaa 69
<210> 45
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 45
tacaatacaa tacaatacaa tacaatacaa tacaatacaa tacaatacaa tacaatacaa 60
tacaataca 69
<210> 46
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 46
ctccactcca ctccactcca ctccactcca ctccactcca ctccactcca ctccactcca 60
ctccactcc 69
<210> 47
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 47
tctaatctaa tctaatctaa tctaatctaa tctaatctaa tctaatctaa tctaatctaa 60
tctaatcta 69
<210> 48
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 48
tagaatagaa tagaatagaa tagaatagaa tagaatagaa tagaatagaa tagaatagaa 60
tagaataga 69
<210> 49
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 49
ggagaggaga ggagaggaga ggagaggaga ggagaggaga ggagaggaga ggagaggaga 60
ggagaggag 69
<210> 50
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 50
ttaaattaaa ttaaattaaa ttaaattaaa ttaaattaaa ttaaattaaa ttaaattaaa 60
ttaaattaa 69
<210> 51
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 51
gccccgcccc gccccgcccc gccccgcccc gccccgcccc gccccgcccc gccccgcccc 60
gccccgccc 69
<210> 52
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 52
gcgcagcgca gcgcagcgca gcgcagcgca gcgcagcgca gcgcagcgca gcgcagcgca 60
gcgcagcgc 69
<210> 53
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 53
ggggagggga ggggagggga ggggagggga ggggagggga ggggagggga ggggagggga 60
ggggagggg 69
<210> 54
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 54
ggcccggccc ggcccggccc ggcccggccc ggcccggccc ggcccggccc ggcccggccc 60
ggcccggcc 69
<210> 55
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 55
tcccatccca tcccatccca tcccatccca tcccatccca tcccatccca tcccatccca 60
tcccatccc 69
<210> 56
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 56
ccccacccca ccccacccca ccccacccca ccccacccca ccccacccca ccccacccca 60
ccccacccc 69
<210> 57
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 57
ccctaaccct aaccctaacc ctaaccctaa ccctaaccct aaccctaacc ctaaccctaa 60
ccctaaccc 69
<210> 58
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 58
cccgaacccg aacccgaacc cgaacccgaa cccgaacccg aacccgaacc cgaacccgaa 60
cccgaaccc 69
<210> 59
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 59
tatacatata catatacata tacatataca tatacatata catatacata tacatataca 60
tatacatat 69
<210> 60
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 60
tcaccatcac catcaccatc accatcacca tcaccatcac catcaccatc accatcacca 60
tcaccatca 69
<210> 61
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 61
ccccaacccc aaccccaacc ccaaccccaa ccccaacccc aaccccaacc ccaaccccaa 60
ccccaaccc 69
<210> 62
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 62
caaaaacaaa aacaaaaaca aaaacaaaaa caaaaacaaa aacaaaaaca aaaacaaaaa 60
caaaaacaa 69
<210> 63
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 63
ccctcaccct caccctcacc ctcaccctca ccctcaccct caccctcacc ctcaccctca 60
ccctcaccc 69
<210> 64
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 64
gcccccgccc ccgcccccgc ccccgccccc gcccccgccc ccgcccccgc ccccgccccc 60
gcccccgcc 69
<210> 65
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 65
gaaaaagaaa aagaaaaaga aaaagaaaaa gaaaaagaaa aagaaaaaga aaaagaaaaa 60
gaaaaagaa 69
<210> 66
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 66
ggagaaggag aaggagaagg agaaggagaa ggagaaggag aaggagaagg agaaggagaa 60
ggagaagga 69
<210> 67
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 67
cccccacccc cacccccacc cccaccccca cccccacccc cacccccacc cccaccccca 60
cccccaccc 69
<210> 68
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 68
taaaaataaa aataaaaata aaaataaaaa taaaaataaa aataaaaata aaaataaaaa 60
taaaaataa 69
<210> 69
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 69
gggagaggga gagggagagg gagagggaga gggagaggga gagggagagg gagagggaga 60
gggagaggg 69
<210> 70
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 70
gccccagccc cagccccagc cccagcccca gccccagccc cagccccagc cccagcccca 60
gccccagcc 69
<210> 71
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 71
gggggagggg gagggggagg gggaggggga gggggagggg gagggggagg gggaggggga 60
gggggaggg 69
<210> 72
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 72
gagacagaga cagagacaga gacagagaca gagacagaga cagagacaga gacagagaca 60
gagacagag 69
<210> 73
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 73
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
ttttttttt 69
<210> 74
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 74
gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 60
ggggggggg 69
<210> 75
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 75
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 60
tgtgtgtgt 69
<210> 76
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 76
tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 60
tatatatat 69
<210> 77
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 77
tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc 60
tctctctct 69
<210> 78
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 78
gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc gcgcgcgcgc 60
gcgcgcgcg 69
<210> 79
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 79
ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc 60
ggcggcggc 69
<210> 80
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 80
ttattattat tattattatt attattatta ttattattat tattattatt attattatta 60
ttattatta 69
<210> 81
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 81
ttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg 60
ttgttgttg 69
<210> 82
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 82
tgctgctgct gctgctgctg ctgctgctgc tgctgctgct gctgctgctg ctgctgctgc 60
tgctgctgc 69
<210> 83
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 83
tagtagtagt agtagtagta gtagtagtag tagtagtagt agtagtagta gtagtagtag 60
tagtagtag 69
<210> 84
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 84
tcctcctcct cctcctcctc ctcctcctcc tcctcctcct cctcctcctc ctcctcctcc 60
tcctcctcc 69
<210> 85
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 85
ttcttcttct tcttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc 60
ttcttcttc 69
<210> 86
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 86
tgatgatgat gatgatgatg atgatgatga tgatgatgat gatgatgatg atgatgatga 60
tgatgatga 69
<210> 87
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 87
tggtggtggt ggtggtggtg gtggtggtgg tggtggtggt ggtggtggtg gtggtggtgg 60
tggtggtgg 69
<210> 88
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 88
atctatctat ctatctatct atctatctat ctatctatct atctatctat ctatctatct 60
atctatcta 69
<210> 89
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 89
gtttgtttgt ttgtttgttt gtttgtttgt ttgtttgttt gtttgtttgt ttgtttgttt 60
gtttgtttg 69
<210> 90
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 90
atttatttat ttatttattt atttatttat ttatttattt atttatttat ttatttattt 60
atttattta 69
<210> 91
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 91
ctttctttct ttctttcttt ctttctttct ttctttcttt ctttctttct ttctttcttt 60
ctttctttc 69
<210> 92
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 92
atggatggat ggatggatgg atggatggat ggatggatgg atggatggat ggatggatgg 60
atggatgga 69
<210> 93
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 93
gttcgttcgt tcgttcgttc gttcgttcgt tcgttcgttc gttcgttcgt tcgttcgttc 60
gttcgttcg 69
<210> 94
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 94
attcattcat tcattcattc attcattcat tcattcattc attcattcat tcattcattc 60
attcattca 69
<210> 95
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 95
gtccgtccgt ccgtccgtcc gtccgtccgt ccgtccgtcc gtccgtccgt ccgtccgtcc 60
gtccgtccg 69
<210> 96
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 96
cttccttcct tccttccttc cttccttcct tccttccttc cttccttcct tccttccttc 60
cttccttcc 69
<210> 97
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 97
atgtatgtat gtatgtatgt atgtatgtat gtatgtatgt atgtatgtat gtatgtatgt 60
atgtatgta 69
<210> 98
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 98
attgattgat tgattgattg attgattgat tgattgattg attgattgat tgattgattg 60
attgattga 69
<210> 99
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 99
ctgtctgtct gtctgtctgt ctgtctgtct gtctgtctgt ctgtctgtct gtctgtctgt 60
ctgtctgtc 69
<210> 100
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 100
gtaggtaggt aggtaggtag gtaggtaggt aggtaggtag gtaggtaggt aggtaggtag 60
gtaggtagg 69
<210> 101
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 101
cgggcgggcg ggcgggcggg cgggcgggcg ggcgggcggg cgggcgggcg ggcgggcggg 60
cgggcgggc 69
<210> 102
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 102
attaattaat taattaatta attaattaat taattaatta attaattaat taattaatta 60
attaattaa 69
<210> 103
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 103
ctccctccct ccctccctcc ctccctccct ccctccctcc ctccctccct ccctccctcc 60
ctccctccc 69
<210> 104
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 104
ctgcctgcct gcctgcctgc ctgcctgcct gcctgcctgc ctgcctgcct gcctgcctgc 60
ctgcctgcc 69
<210> 105
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 105
ctggctggct ggctggctgg ctggctggct ggctggctgg ctggctggct ggctggctgg 60
ctggctggc 69
<210> 106
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 106
tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt 60
tttgttttg 69
<210> 107
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 107
tccattccat tccattccat tccattccat tccattccat tccattccat tccattccat 60
tccattcca 69
<210> 108
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 108
tttattttat tttattttat tttattttat tttattttat tttattttat tttattttat 60
tttatttta 69
<210> 109
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 109
gggctgggct gggctgggct gggctgggct gggctgggct gggctgggct gggctgggct 60
gggctgggc 69
<210> 110
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 110
gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct 60
gagctgagc 69
<210> 111
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 111
tttcttttct tttcttttct tttcttttct tttcttttct tttcttttct tttcttttct 60
tttcttttc 69
<210> 112
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 112
tgtattgtat tgtattgtat tgtattgtat tgtattgtat tgtattgtat tgtattgtat 60
tgtattgta 69
<210> 113
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 113
ggagtggagt ggagtggagt ggagtggagt ggagtggagt ggagtggagt ggagtggagt 60
ggagtggag 69
<210> 114
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 114
tagattagat tagattagat tagattagat tagattagat tagattagat tagattagat 60
tagattaga 69
<210> 115
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 115
tctattctat tctattctat tctattctat tctattctat tctattctat tctattctat 60
tctattcta 69
<210> 116
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 116
ctcctctcct ctcctctcct ctcctctcct ctcctctcct ctcctctcct ctcctctcct 60
ctcctctcc 69
<210> 117
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 117
ttaatttaat ttaatttaat ttaatttaat ttaatttaat ttaatttaat ttaatttaat 60
ttaatttaa 69
<210> 118
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 118
gggcggggcg gggcggggcg gggcggggcg gggcggggcg gggcggggcg gggcggggcg 60
gggcggggc 69
<210> 119
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 119
gcgctgcgct gcgctgcgct gcgctgcgct gcgctgcgct gcgctgcgct gcgctgcgct 60
gcgctgcgc 69
<210> 120
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 120
cccctcccct cccctcccct cccctcccct cccctcccct cccctcccct cccctcccct 60
cccctcccc 69
<210> 121
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 121
ggccgggccg ggccgggccg ggccgggccg ggccgggccg ggccgggccg ggccgggccg 60
ggccgggcc 69
<210> 122
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 122
gggatgggat gggatgggat gggatgggat gggatgggat gggatgggat gggatgggat 60
gggatggga 69
<210> 123
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 123
ggggtggggt ggggtggggt ggggtggggt ggggtggggt ggggtggggt ggggtggggt 60
ggggtgggg 69
<210> 124
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 124
gggttagggt tagggttagg gttagggtta gggttagggt tagggttagg gttagggtta 60
gggttaggg 69
<210> 125
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 125
gggttcgggt tcgggttcgg gttcgggttc gggttcgggt tcgggttcgg gttcgggttc 60
gggttcggg 69
<210> 126
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 126
atatgtatat gtatatgtat atgtatatgt atatgtatat gtatatgtat atgtatatgt 60
atatgtata 69
<210> 127
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 127
tgatggtgat ggtgatggtg atggtgatgg tgatggtgat ggtgatggtg atggtgatgg 60
tgatggtga 69
<210> 128
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 128
gggttggggt tggggttggg gttggggttg gggttggggt tggggttggg gttggggttg 60
gggttgggg 69
<210> 129
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 129
ttgtttttgt ttttgttttt gtttttgttt ttgtttttgt ttttgttttt gtttttgttt 60
ttgtttttg 69
<210> 130
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 130
gggtgagggt gagggtgagg gtgagggtga gggtgagggt gagggtgagg gtgagggtga 60
gggtgaggg 69
<210> 131
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 131
ggcgggggcg ggggcggggg cgggggcggg ggcgggggcg ggggcggggg cgggggcggg 60
ggcgggggc 69
<210> 132
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 132
ttctttttct ttttcttttt ctttttcttt ttctttttct ttttcttttt ctttttcttt 60
ttctttttc 69
<210> 133
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 133
tccttctcct tctccttctc cttctccttc tccttctcct tctccttctc cttctccttc tccttctcc 69
<210> 134
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 134
gggtgggggt gggggtgggg gtgggggtgg gggtgggggt gggggtgggg gtgggggtgg 60
gggtggggg 69
<210> 135
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 135
ttatttttat ttttattttt atttttattt ttatttttat ttttattttt atttttattt 60
ttattttta 69
<210> 136
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 136
ccctctccct ctccctctcc ctctccctct ccctctccct ctccctctcc ctctccctct 60
ccctctccc 69
<210> 137
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 137
ggctggggct ggggctgggg ctggggctgg ggctggggct ggggctgggg ctggggctgg 60
ggctggggc 69
<210> 138
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 138
ccctccccct ccccctcccc ctccccctcc ccctccccct ccccctcccc ctccccctcc 60
ccctccccc 69
<210> 139
<211> 69
<212> DNA
<213> Homo sapiens
<220>
<221> repeat_region
<222> (1)..(69)
<400> 139
ctctgtctct gtctctgtct ctgtctctgt ctctgtctct gtctctgtct ctgtctctgt 60
ctctgtctc 69
<210> 140
<211> 15
<212> DNA
<213> Bacteria
<400> 140
yaacgagcgc aaccc 15
<210> 141
<211> 17
<212> DNA
<213> Bacteria
<400> 141
cggtgtgtac aagaccc 17
<210> 142
<211> 25
<212> DNA
<213> plasmid
<400> 142
ggtaccgtcg acatggctca gcgtc 25
<210> 143
<211> 20
<212> DNA
<213> plasmid
<400> 143
aagcttgtca tcgagctcga 20
<210> 144
<211> 30
<212> DNA
<213> plasmid
<400> 144
tggagaacca cctttcggac cgtgccattc 30
Claims (4)
1. a grand Extraction Methods of Genome, is characterized in that, utilizes probe hybridization magnetic capture mode to reduce the impact of host DNA in host's macro genome DNA sample; Described probe and described host genome DNA sample hybridize rear and described magnetic bead coupling, and concrete steps are as follows:
1) described host's macro genome DNA sample of the described probe and extraction that are marked with vitamin H is hybridized;
2) with the complex body of the described magnetic capture probe and described host DNA that are coated with Streptavidin, and magnetic bead-probe-target DNA mixture is separated with magnetic frame;
3) the magnetic bead scavenging solution after the hybridization solution of removal described magnetic bead-probe-target DNA mixture and hybridization is carried out purifying and reclaim DNA;
Probe 5 ' is terminal modified vitamin H; Described probe is selected from sequence SEQ ID NO:1 to the sequence SEQ ID NO:139 in sequence table.
2. a grand Extraction Methods of Genome, is characterized in that, utilizes probe hybridization magnetic capture mode to reduce the impact of host DNA in host's macro genome DNA sample; With the hybridization of described host genome after described probe and described magnetic bead coupling; Concrete steps are as follows:
1) the described probe conjugate of described magnetic bead and the mark vitamin H being coated with Streptavidin is utilized;
2) by mixture good for coupling and described host's macro genome DNA sample hybridization;
3) remove magnetic bead-probe-target DNA mixture, purifying is carried out to hybridization solution and hybridization scavenging solution and reclaims DNA; Probe 5 ' is terminal modified vitamin H; Described probe is selected from sequence SEQ ID NO:1 to the sequence SEQ ID NO:139 in sequence table.
3. the grand Extraction Methods of Genome according to any one of claim 1 ~ 2, after it is characterized in that hybridization completes, heats or sodium hydroxide method sex change described magnetic bead-probe-target DNA mixture, reclaims the magnetic bead that coupling has probe.
4. the grand Extraction Methods of Genome according to any one of claim 1 ~ 2, is characterized in that described host's macro genome DNA sample takes from human respiratory, oral cavity, digestive tube, skin or reproductive tract.
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| CN103361374A (en) * | 2013-07-23 | 2013-10-23 | 扬州日兴生物科技股份有限公司 | Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene |
| EP3129503A4 (en) * | 2014-04-11 | 2018-02-21 | The Trustees Of The University Of Pennsylvania | Compositions and methods for metagenome biomarker detection |
| WO2016131048A1 (en) * | 2015-02-13 | 2016-08-18 | Icahn School Of Medicine At Mount Sinai | Rna containing compositions and methods of their use |
| CN104789687A (en) * | 2015-05-08 | 2015-07-22 | 上海允英医疗科技有限公司 | Probe preparation method for multi-gene capture sequencing |
| CN106148326A (en) * | 2016-07-27 | 2016-11-23 | 上海美吉生物医药科技有限公司 | The extracting method of macro genome DNA |
| CN111455021A (en) * | 2019-01-18 | 2020-07-28 | 广州微远基因科技有限公司 | Method and kit for removing host DNA in metagenome |
| CN110093409B (en) * | 2019-04-26 | 2020-11-24 | 南京世和基因生物技术股份有限公司 | Infectious line detection method and kit based on high-throughput sequencing |
| CN110205318A (en) * | 2019-05-15 | 2019-09-06 | 杭州杰毅生物技术有限公司 | Macro Extraction Methods of Genome based on CRISPR-Cas removal host genome DNA |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839168A (en) * | 2012-07-31 | 2012-12-26 | 深圳华大基因研究院 | Nucleic acid probe, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102839168A (en) * | 2012-07-31 | 2012-12-26 | 深圳华大基因研究院 | Nucleic acid probe, and preparation method and application thereof |
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| A Human-Specific Subfamily of Alu Sequences;MARK A. BATZER ET AL.;《GENOMICS》;19911231;第481-482页 * |
| Rapid Isolation of DNA Probes within Specific Chromosome Regions by Interspersed Repetitive Sequence Polymerase Chain Reaction;SUSAN A. LEDBETTER ET AL.;《GENOMICS》;19901231;第475-481页 * |
| Subtractive hybridization magnetic bead capture: A new technique for the recovery of full-length ORFs from the metagenome;Quinton C. Meyer et al.;《Biotechnology Journal》;20071231;第36–40页 * |
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