CN101275168B - Array of nucleotidic sequences and method for using this array - Google Patents

Array of nucleotidic sequences and method for using this array Download PDF

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CN101275168B
CN101275168B CN 200810095172 CN200810095172A CN101275168B CN 101275168 B CN101275168 B CN 101275168B CN 200810095172 CN200810095172 CN 200810095172 CN 200810095172 A CN200810095172 A CN 200810095172A CN 101275168 B CN101275168 B CN 101275168B
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gene
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sequence
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dna
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CN101275168A (en
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K·埃伦费尔德斯托尔岑巴赫
J·乌加尔德
A·阿拉韦纳杜阿尔特
N·卢瓦拉
A·马斯塞普尔韦达
P·帕拉达巴尔德坎托斯
R·巴迪利亚奥尔鲍姆
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BIO SIGMA CORP
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Abstract

The invention discloses a nucleotide sequence array that rapidly and synchronously identifies some genes in a microorganism sample. The genes code albumen of related activity in the biological technology and use the method to identify the genes by array. Specifically, genes related to biomembrane forming, CO2 fixing, energy metabolism, chemiotaxis, mobility, iron-oxidization, nitrogen metabolism, oxidation/reduction of albumen of sulfur assimilation and vulcanized compound are coded. In the condition that all needed to appraise microorganism community quality, the nucleotide sequence array is an effective biotechnological tool.

Description

The method of nucleotides sequence column array and this array of use
Invention field
The invention discloses a kind of nucleotides sequence column array for identifying fast and simultaneously that at microbiological specimens some gene exists, the albumen of process related activity in described genes encoding biotechnology, and use this array to identify the method for said gene.Specifically, evaluation is that coding relates to biofilm formation, CO 2Fixing, energy metabolism, chemotaxis and movability, iron oxidation, nitrogen length are thanked, the gene of the albumen of the oxidation/reduction of sulfur assimilation and sulfuration compound.When the quality of needs assessment microflora, the array of this nucleotide sequence is a kind of effective biotechnology instrument.
Background of invention information
Biotechnology typically refers at the biology (microorganism, animal or plant) or their the derivative (meta-bolites that produce or transformation utilizes biosystem (for example complicated microflora), lives for product or the process of specific end use, albumen, nucleic acid) any technology is used.In the present invention, we are absorbed in biotechnology, particularly biomining and the microorganism biological scrubbing of using microorganism.
Many microbial biotechnology processes of the propagation that originally is present in the microflora in pending substrate (be present in ore in biological example mining situation or bioreediation situation under be present in the propagation of the group in contaminated soil or water) have been implemented to strengthen.It will be very useful having in these cases the instrument that can estimate the metabolic characteristic that has group, for example, can judge to utilize the group that exists whether can realize our interested process, perhaps whether we should inoculate other suitable bacterial strains.
Up to now, the method of the most frequently used evaluation microflora is by various technological methods, for example selectivity is cultivated, fluorescence in situ hybridization (FISH), with for example detection probes coupling of specific mark thing, polymerase chain reaction (PCR) and grand and little DNA array are identified the species that exist.In above-mentioned technology, all relate to molecular biological technology, be that FISH, detection probes, PCR and grand and little DNA array are all based on the hybridization of DNA and the known array of unknown sample, described known array has specificity to the microorganism that needs the bolt survey or identify, for example the microorganism of certain kind or genus.
The problem of current method is may exist in these microfloras is permitted eurypalynous different microorganisms, therefore need to carry out many tests determines their composition, then the classification Properties of group of data and each of these acquisitions having been identified associates, and finally determines whether there is interested function in sample.If there is the microorganism of before not describing, even the metabolic activity of this microorganism is known, and be also very important to process, this association can not be implemented.Therefore need a kind of for directly estimating and control microflora metabolic characteristic, rather than for the identification of its method by what the composition of the microorganism.
Method of the present invention is used for having solved this technical problem, the albumen of described genes encoding related activity in biotechnology at the nucleotides sequence column array of microbiological specimens detection and identification code gene and the method for this array of use by foundation.
When the classification of group to be measured was converted to the evaluation that has related activity albumen this group, we directly were absorbed in the essence of bioprocess: the metabolic function of finally realizing these processes with the focus of method.For example, what in each bioleaching process, the existence of searching hook end spirillum (Leptospirillum spp.) was in fact really sought is the iron oxidation, and the every kind microorganism relevant to some bioprocesss is not always the case.
Can the novel method of our design be made correct response to the operator's of biotechnology processes needs: this group's ferric oxide? its sulfur oxide? its fixation of C O 2? its fixed nitrogen? use the method for prior art to answer identical problem, at first the operator should be appreciated that and have which species, and then he or she must carry out each the characteristic in this information and these species related.
In all cases, we are designed for the DNA fragmentation of identification code gene, and described genes encoding has the albumen of related activity in biotechnology, and described fragment meets an essential condition: the specificity of described fragment must unique corresponding target gene.On the other hand, sought as far as possible special and directly to the conservative region of homology (orthologically) to each target gene.This is based on following thought: not only detect the gene that has been sequenced, be not described gene with each target gene homology although also detect those.The result of this retrieval is corresponding to the special fragment of certain gene or function in specific classification group in the specific region.
Utilize the DNA fragmentation of these designs, we develop can the Identifying micro-organisms sample in the DNA fragmentation array that exists of these genes, described genes encoding has the albumen of related activity in biotechnology.
The clearly definition of DNA array is proposed by Schena and its colleague: " permission microcosmic and system's nucleic acid of Analysis of Complex DNA sample is simultaneously arranged " (Schena M., Heller, R.A., Theriault, P., Konrad, K., Lachenmeier, E.and Davis, R.W.Trends Biotechnol.16,301-306,1998).
There is the array of two types in diameter according to the DNA spot of the marking, grand array (300 microns or larger), and microarray (less than 100 microns).The former can prepare by hand in the laboratory, need not just can see spot by special device.The latter needs automatization marking system (normally robot printing platform) and specialized collection and image processing apparatus.
In the present invention, the DNA fragmentation array comprises the marking in flat surface, the orderly serial spot on one deck glass, silicone or nylon for example, and wherein each spot comprises the DNA fragmentation of a large amount of copies, described DNA fragmentation is known, and special for interested certain gene in bioprocess.
Use the hybridization with from the marker DNA extract of testing sample time of " spot " group that the detection method of DNA fragmentation array comprises array.Usually, make the sample DNA experience sex change stage that is labeled and ruptures under particular cases, in this stage, utilize the mode of for example heating that double-stranded DNA is separated.When temperature reduces because physical-chemical characteristic, DNA tend to and with its sequence hybridization of accurate complementation.When this DNA contact array, overlap if exist between the contained DNA fragmentation of sample DNA and spot, marker DNA copy most possible maintenance of sample, be connected with the special of this spot.This comes from and comprises higher complementary DNA copy number in the array spot.The IMAQ of hybridization array and the treatment stage, this mark allows to detect the microorganism that exists in testing sample.
Can utilize any known labeling technique to carry out DNA marker, wherein modal is fluorescence or radio-labeling.
Array and their use are known, and we have found the array example that in the test sample, microorganism exists in the prior art, but they are not absorbed in the detection of gene, the related activity albumen in wherein said genes encoding biotechnology.
The scheme of various disclosed preparation DNA fragmentation arrays is arranged now, and the laboratory that this array preparation service is provided.Therefore, array specificity and validity only depend on the selection of gene and the design of DNA fragmentation used, because in preparation, upholder, DNA fragmentation all can change in conjunction with the spatial distribution of spot on the method for upholder, upholder etc., and this depends on the scheme that adopts in the company that entrusts the array preparation or laboratory itself people such as (, Journal of Microbiological Methods 47 (2001) 257-272) Ye.
The invention summary
The invention discloses a kind of method for identify fast and simultaneously the described gene of this array evaluation of nucleotides sequence column array and use that some gene exists at microbiological specimens, described genes encoding has the albumen of related activity in biotechnology.
We have designed the DNA fragmentation of length 100 or still less nitrogenize base, make the existence of identified gene become possibility, and described genes encoding relates to biofilm formation, CO 2The albumen of the oxidation/reduction of fixing, energy metabolism, chemotaxis and movability, iron oxidation, nitrogen metabolism, sulfur assimilation and sulfuration compound.The fragment of all designs is all special to the gene of the coding specific protein relevant to interested function, so we say that they are special to some metabolic function.In some cases, design oligonucleotides based on gene be only special to a kind, therefore, described oligonucleotide is to function and to plant be all special.Under other consistent zone, find that interested gene is directly conservative to homology.In these cases, design oligonucleotides in above-mentioned straight homologues, therefore described oligonucleotide is special to function and the taxonomical group of discussing.
When having these DNA fragmentations at least a in array, just can estimate the metabolic characteristic of microflora.The array of the DNA fragmentation that comprises several designs preferably is provided, and described array makes simultaneously and the existence of step evaluation several or full gene becomes possibility, and described genes encoding relates to biofilm formation, CO 2The albumen of the oxidation/reduction of fixing, energy metabolism, chemotaxis, nitrogen metabolism, sulfur assimilation and sulfuration compound.
When the quality needs assessment of the microflora that relates to specific metabolic function with when controlling, the method is the effective tool in biotechnology.
The accompanying drawing summary
Fig. 1 is corresponding to the result of biomining sample 1 with microarray hybridization of the present invention.Fragment of the present invention is corresponding to the sub-fragment of the described fragment of table 2.Describe the content of each position of microarray in table 1 in detail, table 3 has been summarized the positive or the negative findings of hybridization.
Result shows the existence of gene, and described genes encoding relates to the albumen of following function: metabolism, the CO of sulfuration compound 2Fixing, iron oxidation, nitrogen are fixed, and are related to the energy metabolism key enzyme.As if sample do not contain the gene about chemotaxis and biofilm formation of evaluation.Positive control shows hybridization signal, and negative control is not labeled yet.
Fig. 2 is corresponding to the result of the second biomining sample and microarray hybridization of the present invention.The microarray that uses is identical with sample 1 use, and its content description is in table 1.The fragment of using in this example is included in the sequence of table 2 description, and table 4 has been summarized the positive or the negative findings of hybridization.
Result shows the existence of gene, and described genes encoding relates to the albumen of all identified functions-so its mixed culture seemingly.Different from previous sample, we observe the height performance of the gene that relates to signaling and chemotaxis and biofilm formation.Positive control shows hybridization signal, and negative control is not labeled yet.
Detailed Description Of The Invention
The invention provides a kind of method of array for and simultaneously identified gene existence quick at microbiological specimens and this array of use, described genes encoding has the albumen of related activity in biotechnology, and described array can be used for the every field of industry and commerce.For example, estimate bioprocess substrate (biological example mining process ore; Bioreediation process contaminated soil or water are perhaps in any other material in biotechnology applications) the wild microflora that exists.Second aspect is that it allows to control the microflora that relates to the biotechnology processing.
In order to satisfy the needs of these biotechnology industry and commerce, we have designed the gene specific DNA fragmentation of length 100 or still less hydrogenation base, and described genes encoding relates to biofilm formation, CO 2The albumen of the oxidation/reduction of fixing, energy metabolism, chemotaxis and movability, iron oxidation, nitrogen metabolism, sulfur assimilation and sulfuration compound.The fragment that these are distributed in the DNA array makes the existence of identifying these genes in sample become possibility, and described sample is from wild or microflora that related to the biotechnology processing.
The fragment of all designs is all special to gene, and described genes encoding relates to the differential protein of interested function, so we say that they are special to some metabolic function, specifically to following function: biofilm formation, CO 2The oxidation/reduction of fixing, energy metabolism, chemotaxis and movability, iron oxidation, nitrogen metabolism, sulfur assimilation and sulfuration compound is.
In some cases, design oligonucleotides based on gene be only special to a kind, therefore, described oligonucleotide is to function and to plant be all special.In other cases, find that interested gene is directly conservative to homology in certain fixed different plant species that belongs to.In these cases, above-mentioned straight to the consistent zone design oligonucleotide between homology, therefore described oligonucleotide is special to function and the taxonomical group of discussing.
How to develop this method in order to understand better us, we will simply describe the step of employing:
At first, we have determined interested function in biotechnology that we want to detect, and the key gene of these functions.
Figure S2008100951726D00052
Secondly, select to have the microorganism of the gene that relates to function interested, described microorganism should meet two primary conditions, has any interested function on the one hand, on the other hand, is completely or partially checked order.
Figure S2008100951726D00053
Again, produce the list of reference sequences, the known microorganism of described sequence encoding, for example interested albumen or function in intestinal bacteria (E.coli), and check homology between described known and interested genome.In the situation that find homology, limit " homology " district corresponding to the gene of interest in microorganism interested.
Figure S2008100951726D00054
Obtain to comprise by this method the initial data storehouse of the various gene of interest sequences in various microorganisms interested.Obviously, not all interested gene all is present in all interested microorganisms (this is the genome of known microorganism/ checked order).
Figure S2008100951726D00055
All genes of the gene order that comprises in the initial data storehouse and microorganism interested compare, and only select the zone special to gene of interest, and in other words, this zone does not show homology with the gene with other functions.The searching algorithm of having inputted these restriction sundries by employing realizes this selection.
The special zone of selecting is like this sorted out by high similarity, and described high similarity can be interpreted as about 80% consistence of the final lengths of designed oligonucleotide, its objective is and detects possible consistent zone (consensus region).
Figure S2008100951726D00062
Consider the physical-chemical variable (GC forms level, hybridization temperature, possible secondary structure) of oligonucleotide, these special zones be confirmed to be for detection of the zone.The zone that meets these conditions is considered to the oligonucleotide material standed for.
Figure S2008100951726D00063
Oligonucleotide material standed for and all known sequences be homology relatively, gets rid of the interested gene with obvious homology from relatively, and wherein said oligonucleotide is based on described gene design.
We choose special oligonucleotide for detection of interested function.
As noted earlier, the first step is to select interested function.During we have selected to be present in microflora in the DNA array and in biotechnology useful 8 metabolism kinetic energy: biofilm formation, CO 2The oxidation/reduction of fixing, energy metabolism, chemotaxis and movability, iron oxidation, nitrogen metabolism, sulfur assimilation and sulfuration compound.Their importance is all apparent concerning any technician who understands thoroughly this technology.
Some in these functions provide general and relevant information about any microflora to us, and for example, whether energy metabolism and group can or can not fixation of C O 2Perhaps nitrogen is to set up the required nutriment of group relevant.Understand the biofilm formation ability of group, the existence of the microorganism that perhaps can move in group, to want the culture condition of the biotechnology processes realized be important equally for setting up us.On the other hand, the oxidation/reduction process of iron oxidation, sulfur assimilation and sulfuration compound in very important particular organisms technical finesse, is also very important process in biological example mining and bioreediation.
Secondly, determine design oligonucleotides based on interested microorganism, described oligonucleotide is included in microarray.Consider two related fields during selection: at first, microorganism has at least a interested function, and secondly, their genome is completely or partially checked order.Consider these factors, select bacterial strain, described bacterial strain has the character of Biosigma:Wenelen (DSM 16786) and Licanantay (DSM 17318), has one or more described metabolic functions, and has the genome that has checked order.Also comprise and have iron and 23270 kinds of thiobacillus ferrooxidant (Acidithiobacillusferrooxidans) ATCC that vulcanizes the compound oxidation function.Two kinds of open sequences are used for Pseudomonas Pseudomallei (Burkholderia pseudomallei) and plant: Pseudomonas Pseudomallei 1710b and Pseudomonas Pseudomallei K96243.Select desulfovibrio desulfurican (Desulfovibrio desulfuricans) to be used for vulcanization-oxidization active, and use disclosed desulfovibrio desulfurican G20 series; Also comprise thiobacillus denitrificans (Thiobacillusdenitrificans) ATCC 25259, its function is the sulfuration compound oxidation.also comprise biomining microorganism Ferroplasma, Leptospirillum (Leptospirillum), sulfolobus solfataricus belongs to (Sulfolobus) and Thermoplasma (Thermoplasma), wherein disclosed sequence is corresponding to following microorganism: Ferroplasmaacidarmanus, Ferroplasma sp. (II), Leptospirillum sp. (II), Leptospirillumsp. (III), sulfolobus acidocaldarius (Sulfolobus acidocaldarius) DSM639, sulfolobus solfataricus (Sulfolobus solfataricus), super hyperthermophilic archaeon strain (Sulfolobus tokodaii), thermoplasma acidophilum (Thermoplasma acidophilum) and hot volcanic substance (Thermoplasmavolcanium).
In case what clear and definite interested function and the microorganism that we will study be, we just can determine to adopt what gene to identify the existence of described function.The below has listed various interested functions and has selected to be used for to represent the summary of their gene.
The description of function interested
The iron oxidation.Fe + 2To Fe + 3The iron oxidation be the essential step of bioleaching process progress because the iron (III) that produces is strong oxidizer, the sulfide or any compound that needs oxidation that can oxidation exist, thereby discharge interested metal in solution.
In order to identify the existence of " iron oxidation " function, select the gene of this process associated protein of following coding based on thiobacillus ferrooxidant (A.ferrooxidan):
rus,cyc1,cyc2,cycA?y?cycA2
In microorganism interested (known microorganisms/ checked order genome), the definition of all these gene orders all can obtain, and therefore need not to use reference sequences.
Biofilm formation.The ability of formation microbial film or silt is all very important for the propagation of any bacterial flora, because it guarantees to stick to the persistence of the biomass on upholder.
In order to identify the existence of " biofilm formation " function, select the gene of this process associated protein of following coding:
galE,galU,rfbA,rfbB,rfbC,rfbD,epsD,gdh,manB,manC,pgm,uppS,wza,wzc
Can not obtain the information about these genes in microorganism interested, so need to according to the homology between the reference gene described gene of searching and microbial genome interested, exist with " possibility " of determining these genes.In most cases, colibacillary gene is used as the reference of other genes except gene epsD, and lactobacillus lactis (Lactobacillus lactis) is as the reference of epsD.
CO 2Fixing.CO 2It is important being fixed in the microbial biotechnology processing, because its allows to estimate whether group is autotrophy, and this can give this processing economic advantages, adds the carbonization substrate because need not in group.
In order to identify " CO 2Fixing " existence of function, select the gene of this process associated protein of following coding:
cbbL1,cbbL2,cbbM,cbbS1,cbbS2,cbbArchea,ppc,prk
Can only obtain now about some in these genes of microorganism interested i.e. cbbL1, cbbL2, cbbM, the information of cbbS1 y cbbS2.In other cases, carry out work according to the reference gene, seek these with reference to the homology between gene and microbial genome interested, exist with " possibility " of determining these genes.Methanosarcina acetivorans is used as the reference of the gene cbb of archeobacteria (Archaeas), and colibacillary gene is used as the reference of gene ppc, and the gene of poly-coccus (Synechococcus) is used as the reference of gene prk.
Chemotaxis and movability.The microorganism movability comes from the existence of flagellum; Under particular cases, mobile coming from training replying of Metabolites Concentration in base, this is called as chemotaxis.After having understood microflora and whether having chemotaxis and ambulant character, just might process in better mode as biotechnology leaved for development and set up culture condition.
In order to identify the existence of " chemotaxis and movability " function, select the gene of this process associated protein of following coding:
flhA,flhB,fliF,fliG,fligM,fliN,motA,motB
Can not obtain the information about any these genes of interested microorganism, so carry out work according to the reference gene, seek these with reference to the homology between gene and microbial genome interested, exist with " possibility " of determining described gene.In each situation, colibacillary gene is used as reference.
Nitrogen metabolism.About the metabolism of nitride compound, there are two kinds and are considered to important process.A kind of in them is that nitrogen is fixed, and its essentially consist is nitrogenase.Another kind is the degraded of the nitride compound of nitrate reductase participation.
In order to identify the existence of " nitrogen metabolism " function, select the gene of this process associated protein of following coding:
nifK,narH,nirA
Can not obtain the information about these genes of microorganism interested, so carry out work according to the reference gene, seek these with reference to the homology between gene and microbial genome interested, exist with " possibility " of determining these genes.Colibacillary gene is used as the reference of narH; Azotobacter vinelandii (Azotobacter vinelandii) is used as the reference of nifK, and poly-coccus (Synechococcus) is used as the reference of nirA.
Sulfur assimilation.Sulfur assimilation is a kind of sulphur in environment to be fixed as organosulfur to be used for the process of cellular metabolism.Two kinds of main end products of this process are indispensable amino acid halfcystine and methionine(Met).
In order to identify the existence of " sulfur assimilation " function, select the gene of this process associated protein of following coding:
cysl,cysJ
Can not obtain the information about these genes of microorganism interested, so carry out work according to the reference gene, seek these with reference to the homology between gene and microbial genome interested, exist with " possibility " of determining described gene.Colibacillary gene is used as the reference of gene cysl and cysJ.
The oxidation/reduction of sulfuration compound.With the process of bioleaching height correlation be the oxidation of sulphur compound.For example, the microorganism that acidophilic bacteria (acidithiobacillus) belongs to can utilize oxygen as electron acceptor(EA), the sulphur compound that catalysis is reduced (sulfide for example, elementary sulfur, sulphur hydrochlorate (thionate), etc.) oxidation, produce end product sulfuric acid, and the reduction kinds such as sulphite and thiosulphate be intermediate product, this makes the dissolving of the metal of being combined with sulfide in ore become possibility.
In order to identify the existence of " oxidation/reduction of sulfuration compound " function, select the gene of this process associated protein of following coding:
doxA,doxD,doxDA1,doxDA2,dsrA,dsrB,dsrC,dsrE,dsrF,dsrK,dsrL,dsrM,dsrN,dsrO,dsrP,dsrS,soxB
Only obtained the doxDA1 of microorganism interested, the information of doxDA2 gene.In other cases, carry out work according to the reference gene, seek these with reference to the homology between gene and microbial genome interested, exist with " possibility " of determining these genes.Acidarmanus Ambivalens is used as gene doxA, the reference of doxD, the gene of Paracoccus denitrificans (Paracoccus denitrificans) is used as the reference of gene soxB, and the gene of Allochromatium vinosum is used as the reference of all dsr genes.
Energy metabolism.
In order to identify the existence of " energy metabolism " function, select the gene of this process associated protein of following coding:
pfk,korA,korB,idh,pdhA,pdhB,pdhC,gltA,accA,accB,aaC,accD
Only obtained the information of the pdhC gene of microorganism interested.In other cases, carry out work according to the reference gene, seek these with reference to the homology between gene and microbial genome interested, exist with " possibility " of determining these genes.Colibacillary gene is used as gene pfk, idh, gltA, accA, accB, the reference of aaC and accD.Methanocaldococcus jannaschii is used as the reference of gene korA and korB, and mycobacterium tuberculosis (Mycobacterium tuberculosis) is used as the reference of gene pdhA and pdhB.
Disclosed described genes encoding has the albumen of related activity in biotechnology for detecting at microbiological specimens and the nucleotides sequence column array of identified gene, comprises a kind of, several or all representatives in the following DNA fragmentation that is attached to its surface:
A. the DNA fragmentation of the gene of at least a special identification code biofilm formation associated protein;
B. at least a special identification code CO 2The fixing DNA fragmentation of the gene of associated protein;
C. the DNA fragmentation of the gene of at least a special identification code energy metabolism associated protein;
D. the DNA fragmentation of the gene of at least a special identification code chemotaxis and movability associated protein;
E. the DNA fragmentation of the gene of at least a special identification code iron oxidation associated protein;
F. the DNA fragmentation of the gene of at least a special identification code nitrogen metabolism associated protein;
G. the DNA fragmentation of the gene of at least a special identification code sulfur assimilation associated protein;
H. at least a special identification code vulcanizes the DNA fragmentation of the gene of compound oxidation/reduction associated protein;
Wherein all there are hundreds of copies in every kind of DNA fragmentation, forms the spot of homology component, and solid is distributed in support surface.
We design long 80 to 100 special nitrogenize bases for each these said gene, and the fragment of preferred 100 special nitrogenize bases, these fragments are open in sequence table.
We have designed altogether 232 DNA fragmentations for the identification of gene, and described genes encoding has the albumen of related activity in biotechnology, 100 Nucleotide of all sheet segment lengths.The sequence of 232 DNA fragmentations of design is included in sequence table.
In 232 sequences of design, sequence number 1 to 86 is special to the biofilm formation function.
Sequence 87 to 101 is to CO 2Fixed function is special.
Sequence 102 to 156 is special to the energy metabolism function.
Sequence 157 to 192 is special to chemotaxis and mobility functions.
Sequence number 193 to 197 is special to the iron oxidative function.
Sequence number 198 to 203 is special to the nitrogen metabolism function.
Sequence number 204 to 210 is special to the sulfur assimilation function.
Special to sulfuration compound oxidation function with sequence 211 to 232.
The fragment of design can be printed in array, and it can be complete or comprise its more long segment, the fragment that the anyon fragment part that perhaps comprises to fragment is similar, the perhaps reverse complementary sequence of all above-mentioned options.Can print easily the sub-fragment of 50 to 70 Nucleotide.
Need to keep the array that the present invention includes firmly in mind is at least a array that is included in the DNA fragmentation of sequence number 1 to 232, and no matter this fragment is complete; Described fragment is present in larger zone and representative surpasses this larger zone (just as the PCR product) of 20%; Or part as being contained in one of sub-fragment in each fragment as disclosed herein, the perhaps reverse complementary sequence of all above-mentioned options.
Above-mentioned aspect is extremely important, because the specificity of nucleotide sequence is identical with its reverse complementary sequence.This specific character, namely specificity is the most inaccessible in the design dna fragment.Utilize existing a series of instruments in prior art although understand thoroughly the personnel of this technology, can distinguish Thermodynamically stable and unsettled oligonucleotide, but still the situation that the stability of complementary sequence is not suitable for using might occur in array.All reverse complementary sequences of fragment number 1 to 232 of the present invention all are considered to belong to scope of the present invention, namely no matter described fragment is complete; Described fragment is present in larger zone and representative surpasses 20% this larger zone, (just as the PCR product); Or part as being contained in one of sub-fragment in each fragment as disclosed herein.
The sub-fragment that preferentially comprises at least a fragment or every kind of interested fragment for array.Also might prepare the array that comprises all disclosed DNA fragmentations or sub-fragment, perhaps under particular cases, only comprise wherein a kind of array.All these options, and all possible intermediate combination, all within the scope of the present invention.
The validity of array of the present invention depends on specificity and the stability for the treatment of marking fragment.These characteristics continue in each sub-fragment contained in designed fragment.This means if use Nucleotide 1 to 100, perhaps 42 to 92 or 15 to 65 or other possible selections arbitrarily, specificity is identical.Every kind of selection is corresponding sub-fragment all, and comprises within the scope of the invention.
Also might be the fragment of the present invention of other oligonucleotide or the DNA fragmentation of sub-fragment by synthesizing or obtaining to comprise the survey wing as the PCR product.The larger zone that comprises current open fragment is also included within scope of the present invention, and the specificity in wherein said larger zone is that the present invention designs with disclosed fragment or sub-fragment and gives.
In order to realize array of the present invention, the fragment of every kind of selection or sub-fragment must be synthesized hundreds of copies, on the suitable upholder of array, the marking (as the homology spot) on existing other materials arbitrarily in glass, silicone, nylon or this area for example.
We point out when background information of the present invention is discussed, and DNA fragmentation synthesizes and the array technology of preparing is known, and they all can be used for preparing array of the present invention.
Except these specific DNA fragments for the gene of the related activity albumen in the encoding human technology, can comprise feminine gender and positive control at each array very easily.The nucleotide sequence that negative control should corresponding never can be found in the biotechnology content.The nucleotide sequence that positive control should corresponding always exist in the problem sample.
Expert for this area, the DNA fragmentation that it is evident that 232 designs of the present invention uses in array, when using, can both identification code has the gene of the related activity albumen in biotechnology in other Protocols in Molecular Biologies based on the hybridization of unknown DNA and specific fragment.In these Protocols in Molecular Biologies, we can mention for example detection probes, FISH, PCR and order-checking.
Utilize 232 DNA fragmentations disclosed by the invention in any Protocols in Molecular Biology, all be considered to obvious application of the present disclosure, belong to scope of the present invention, and no matter described fragment is complete; Described fragment is present in larger zone and representative surpasses this larger zone of 20%, (just as the PCR product); Or part as being contained in one of sub-fragment in each fragment as disclosed herein; The perhaps reverse complementary sequence of all above-mentioned options.
The purposes of array
In order to utilize array of the present invention to detect and identify the existence of some gene in microbiological specimens, at first the albumen of the related activity in described genes encoding biotechnology need to separate our sample DNA of estimating interested.Also might utilize cDNA, that wherein unique difference is that the fs separates in this case is sample RNA.In the art, the different modes that extracts DNA and RNA from environmental sample or culture is known, can use according to the special properties of sample in every kind of situation any one in them.
Subordinate phase, the DNA of all samples or RNA must be converted to short labeled dna fragment, described fragment be fit to the array spot in the fragment hybridization of the marking.If sample DNA is separated, it must be by fragmentation and mark.When not utilizing the RNA of sample, fragmentation is optional, only need carry out markers step to obtain the cDNA of mark.A kind of can fragmentation and the technology of marker DNA be to speculate beam splitter (aleatory splitters) mark with 6 Nucleotide of DNA.Utilize the existing any technology in this area, for example radioactivity, vitamin H, fluorescence or other marks, use be labeled or the Nucleotide that is easy to mark is carried out mark.If use grand array, mark preferably uses radio-labeling, 32P, if use microarray, will use fluorescent mark, for example, utilizes Cy5 or Cy3.
Describedly be not limited to the present invention for the preparation of the DNA of array or the method for cDNA, can use existing DNA or cDNA preparation method in any prior art, this use that does not represent array can depart from the scope of the present invention.
In case DNA is produced, it is placed in the DNA sex change stage.Place the DNA mixture of decile on array, then the DNA of this sex change is hatching, is allowing array hybridize at least one hour, the hybridization of preferably spending the night in the temperature range of 40 to 70 ℃ on array.
After hybridization, carefully wash array.Usually at the temperature of gentleness, for example 35-50 ℃, preferred 40-45 ℃, utilize tenderising soln (softening solutions) to wash.
In case array is washed, it preferably is dried by centrifugal easily, and for example, in Falcon pipe or some analogues, short period of time and moderate speed (every 1 to 5 minute of 200-3000xg) are centrifugal.
At last, need to manifest which spot show tags.The existence of the position indicator of each mark spot, described genes encoding has the albumen of related activity in biotechnology, and DNA fragmentation is based on described gene design.
Need to check that the spot corresponding to negative control is not labeled yet, because if the hybridization of existence and these DNA fragmentations, it shows that this reaction is non-specific, and institute's result that obtains must be by reject, because they may comprise false positive.
The spot of corresponding positive control should be labeled equally, because when not occuring with the hybridization of this DNA fragmentation, it shows to exist in reaction disturbs, and therefore there is no the corresponding false negative of spot possibility of signal.
According to foregoing, the existence of determining some gene in microflora will be reduced to reading of mark spot in array of the present invention, and described genes encoding has the albumen of related activity in biotechnology.
Embodiment
Embodiment 1: detect and identify the microarray that some gene exists, described genes encoding has the albumen of the related activity in biotechnology.
Preparation has the microarray of 23 different DNA fragmentations of the present invention, and described microarray identification code has the gene of related activity albumen in biotechnology, and wherein these are active relevant to following function: sulfur assimilation, CO 2Fixing, iron oxidation, nitrogen metabolism, energy metabolism, chemotaxis and movability, and biofilm formation.
Also comprise two positive controls and two negative controls in microarray.The detailed description of each position of this microarray is in following table 1.
Table 1
Function Position in microarray
Sulfur assimilation A1-A3
CO 2Fixing A4-A6
The iron oxidation A7-A9
Nitrogen metabolism B2-B4
Energy metabolism B5-B7
Chemotaxis and movability B8,C1-C3
Biofilm formation C4-C7
Negative control B9,C8
Positive control B1,C9
The fragment of all markings is all 60 Nucleotide.Selected DNA fragmentation of the present invention is corresponding to the sub-fragment of 60 oligonucleotide of fragment shown in table 2, and described fragment defines in sequence table.
Table 2
Function Position in microarray Sequence number
Sulfur assimilation A1-A3 208-210
CO 2Fixing A4-A6 92-94
The iron oxidation A7-A9 193-195
Nitrogen metabolism B2-B4 201-203
Energy metabolism B5-B7 113-115
Chemotaxis and movability B8,C1-C3 167-170
Biofilm formation C4-C7 2-5
Negative control B9,C8 ?
Positive control B1,C9 ?
Trust provides the specialized company of this service to prepare microarray.
Embodiment 2: microarray for detection of with the purposes of identified gene, described genes encoding has the albumen of the related activity in biotechnology.
Embodiment 1 microarray that obtains is used for determining two bioleachings accumulation waste liquid samples, the i.e. compositions of microflora in sample 1 (S1) and sample 2 (S2).
Utilize the total DNA in conventional DNA extraction method extraction S1 and S2.
From sample extraction 2 μ l DNA, be placed in the Eppendorf pipe.Follow in each case following method:
Add 36 μ l ddH 2O and 3.3mL commercialization 6 Nucleotide (six aggressiveness) are speculated beam splitter (aleatorysplitters).Boiled 5 minutes, and then be placed in ice.
Add 2 μ l mixture of ribonucleotides, wherein Nucleotide dUTP fluorophore Cy mark.The Cy5 that shows red fluorescence is used for S1, and shows that the Cy3 of green fluorescence is used for S2.After this, add the buffered soln (according to the supplier) that 4 μ l Klenow type polysaccharases and 5 μ l are suitable for polysaccharase; Hatched 4 hours at 37 ℃.
With 5 μ l AEDT 0.5M pH8 termination reactions.Use ion exchange column to reclaim the DNA of mark.Vacuum-drying comprises the elutriant of DNA.
Add 100 μ l Tris pH 7 buffered soln as alkaline components, resuspended DNA, and be placed in 100 ℃ lower 1.5 minutes so that the DNA sex change.Hybridization is completed in vibration whole night under on microarray 55 ℃.
Morning, each microarray at 45 ℃ with 2 * SSC, 0.1%SDS (sodium lauryl sulphate) washed twice; With 0.2 * SSC, 0.1%SDS washs once at 42 ℃, at 42 ℃ with 0.2 * SSC washing once.(for 500 SSC 20 *, 87.6g NaCl, 50g Trisodium Citrate (2H 2O), adjust to pH 7.0).
Each array is placed in the box 15 minutes that fills miliQ water, then in Falcon pipe or some analogues with 1.100rpm centrifugal drying one minute.
Might observe the result of microarray at last, Fig. 1 shows the S1 result, and Fig. 2 shows the S2 result.
In table 3, indicated the position of different fragments in the microarray, and that summarized that Fig. 1 shows and result available from the DNA hybridization of S1.All positive controls all show hybridization, and negative control is not labeled yet.
Table 3, sample 1s (1)
Function Position in microarray Result
Sulfur assimilation A1 A2 A3 - - +
CO 2Fixing A4 A5 A6 + + +
The iron oxidation A7 A8 A9 + - +
Nitrogen metabolism B2 B3 B4 + - -
Energy metabolism B5 B6 B7 + - -
Chemotaxis and movability B8 C1 C2 C3 - - - -
Biofilm formation C4 C5 C6 C7 - - - -
Negative control B9 C8 - -
Positive control B1 C9 + +
Mark: (+): the positive; (-): feminine gender.
Result shows the existence of gene, and described genes encoding relates to sulfur assimilation, CO 2The albumen of fixing, iron oxidation and nitrogen fixed function, and the key protein that relates to energy metabolism.As if this sample do not contain the gene that relates to chemotaxis and biofilm formation of evaluation.
In table 4, the position of different fragments in display microarray again, and general introduction Fig. 2 that show with result available from the DNA hybridization of S2.All positive controls all show hybridization, and negative control is not labeled yet.
Table 4, sample 2s (2)
Function Position in microarray Result
Sulfur assimilation A1 A2 A3 + + -
CO 2Fixing A4 A5 A6 - + +
The iron oxidation A7 A8 A9 + + -
Nitrogen metabolism B2 B3 B4 + + +
Energy metabolism B5 B6 B7 + + -
Chemotaxis and movability B8 C1 C2 C3 + + + +
Biofilm formation C4 C5 C6 C7 + + + +
Negative control B9 C8 - -
Positive control B1 C9 + +
Mark: (+): the positive; (-): feminine gender.
Result shows the existence of gene, and described genes encoding relates to the albumen of all Function of Evaluation.This is the example of mixed culture obviously.Different from previous sample, we observe the height performance of signaling and chemotaxis and the biofilm formation of cell.
The present embodiment is for characterization, should not be considered to limitation of the scope of the invention, and scope of the present invention limits in appended claims.
Sequence table
Figure S2008100951726D00191
Figure S2008100951726D00201
Figure S2008100951726D00211
Figure S2008100951726D00221
Figure S2008100951726D00231
Figure S2008100951726D00241
Figure S2008100951726D00251
Figure S2008100951726D00261
Figure S2008100951726D00271
Figure S2008100951726D00281
Figure S2008100951726D00291
Figure S2008100951726D00301
Figure S2008100951726D00311
Figure S2008100951726D00331
Figure S2008100951726D00341
Figure S2008100951726D00351
Figure S2008100951726D00371
Figure S2008100951726D00381
Figure S2008100951726D00401
Figure S2008100951726D00411
Figure S2008100951726D00421
Figure S2008100951726D00431
Figure S2008100951726D00441
Figure S2008100951726D00451
Figure S2008100951726D00461
Figure S2008100951726D00471
Figure S2008100951726D00481
Figure S2008100951726D00491
Figure S2008100951726D00501
Figure S2008100951726D00511
Figure S2008100951726D00521
Figure S2008100951726D00531
Figure S2008100951726D00561
Figure S2008100951726D00571
Figure S2008100951726D00591
Figure S2008100951726D00621
Figure S2008100951726D00641
Figure S2008100951726D00651
Figure S2008100951726D00661
Figure S2008100951726D00671
Figure S2008100951726D00681
Figure S2008100951726D00691
Figure S2008100951726D00711
Figure S2008100951726D00721
Figure S2008100951726D00731
Figure S2008100951726D00741
Figure S2008100951726D00751
Figure S2008100951726D00761
Figure S2008100951726D00771
Figure S2008100951726D00781
Figure S2008100951726D00791
Figure S2008100951726D00801
Figure S2008100951726D00811
Figure S2008100951726D00821
Figure S2008100951726D00831
Figure S2008100951726D00841
Figure S2008100951726D00861
Figure S2008100951726D00871
Figure S2008100951726D00881
Figure S2008100951726D00901
Figure S2008100951726D00911
Figure S2008100951726D00921
Figure S2008100951726D00931
Figure S2008100951726D00941
Figure S2008100951726D00951
Figure S2008100951726D00971
Figure S2008100951726D00981
Figure S2008100951726D00991
Figure S2008100951726D01001
Figure S2008100951726D01011
Figure S2008100951726D01021
Figure S2008100951726D01031
Figure S2008100951726D01061
Figure S2008100951726D01071
Figure S2008100951726D01081
Figure S2008100951726D01091
Figure S2008100951726D01101
Figure S2008100951726D01121
Figure S2008100951726D01131
Figure S2008100951726D01161
Figure S2008100951726D01171
Figure S2008100951726D01181
Figure S2008100951726D01191
Figure S2008100951726D01201
Figure S2008100951726D01211
Figure S2008100951726D01221
Figure S2008100951726D01231
Figure S2008100951726D01241

Claims (4)

1. be used for the nucleotides sequence column array at the gene of microbiological specimens detection and identification code albumen, it is characterized in that this array comprises
By sequence number 25 or its reverse complementary sequence, or at least a DNA fragmentation of the sub-fragment of 50 to 70 Nucleotide of sequence number 25 or its reverse complementary sequence composition, its specific binding biofilm formation gene; With
Be attached at least a DNA fragmentation of array surface, wherein said DNA fragmentation is selected from the sub-fragment of following sequence or its 50 to 70 Nucleotide:
A. sequence number 1 to 86 and their reverse complementary sequence, be specific to the biofilm formation gene;
B. sequence number 87 to 101 and their reverse complementary sequence, be specific to CO 2Fixed base because of;
C. sequence number 102 to 156 and their reverse complementary sequence, be specific to the energy metabolism gene;
D. sequence number 157 to 192 and their reverse complementary sequence, be specific to chemotaxis and movability gene;
E. sequence number 193 to 197 and their reverse complementary sequence, be specific to the iron oxidoreductase gene;
F. sequence number 198 to 203 and their reverse complementary sequence, be specific to the nitrogen metabolism gene;
G. sequence number 204 to 210 and their reverse complementary sequence, be specific to the sulfur assimilation gene; Or
H. sequence number 211 to 232 and their reverse complementary sequence are specific to sulfuration compound oxidation/go back protogene;
Wherein all there are hundreds of copies in every kind of DNA fragmentation, form the spot of homology component, and solid is distributed in array surface.
2. array as claimed in claim 1, is characterized in that described DNA fragmentation is comprised of 50-70 Nucleotide.
3. detect the method for the gene of proteins encoded in microbiological specimens, it is characterized in that comprising:
A. hatch the nucleic acid samples available from microbiological specimens on array claimed in claim 1,
B. after hatching, the washing array to be removing not the labeled dna fragment of hybridization, and
C. the pair array spot develops.
4. the method for the gene of identification code albumen in microbiological specimens is characterized in that comprising:
A. extract nucleic acid from microbiological specimens; And/or utilize be labeled or Nucleotide that be easy to mark, nucleic acid samples is carried out fragmentation and labeling nucleic acid sample;
B. hatch the nucleic acid samples available from microbiological specimens on array claimed in claim 1;
C. after hatching, the washing array to be removing not the labeled dna fragment of hybridization, and
D. the pair array spot develops.
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