CN101034061A - Method for detecting mononucleotide polymorphism with biochip - Google Patents
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Abstract
This invention relates to a method of biochip detecting mononucleotide polymorphism. first amplify nucleic acid sample, obtain amplifying product; then take solution hybridization and joining enzyme reaction, connect complete matching probe in solution hybridization with currency probe, to detect mononucleotide polymorphism; then carry out solid-phase hybridization, make label probe with 5' end band, probe 3's end with detecting base group and biochip to carry out hybridization, temperature 25 - 75deg, time 0.5h to 36h, detecting base group be distributed to different position of biochip; Carry out mononucleotide polymorphism detecting, gain detecting outcome. This invention solves technical problems of background technology hard of realizing high flux reactor detecting and high detecting cost.
Description
Technical field
The invention belongs to a kind of field relevant, be applicable to that the nucleotide of specific site on the nucleic acid molecules is differentiated, be specifically related to a kind of method of detecting mononucleotide polymorphism with biochip with genetic test.
Background technology
Calendar year 2001 single nucleotide polymorphism cooperative groups (The SNP Consortium) has made up and has contained 1.42 * 10
6Individual SNP, density reach human inheritance's collection of illustrative plates that every 1.9kb fragment contains a mononucleotide polymorphism site, and this is extremely important to the research of carrying out Disease-causing gene and origin of mankind and evolution.According to estimates, nearly 100,000 SNP molecular labelings will be used to gene function and with the research of disease association.So huge analyze demands has proposed high requirement to detection technique.
At present, the common detection method for allelic single nucleotide polymorphism mainly contains following several:
The allele-specific amplification method is the sequence complementation that makes 3 of designed primer a 1 ' end and an equipotential P gene to be measured in the system of PCR, and not complementary with the sequence of another allele (Q gene); When polymerase chain reaction (PCR) amplification, amplify the P gene.According to this, the sequence complementation of design primer 2 and Q gene is carried out electrophoresis with amplified production respectively again.Though this method can detect single nucleotide polymorphism, complex operation step is had relatively high expectations to design of primers, and therefore, operation easier and workload are bigger.
Two-way specific allele pcr amplification method, identical with allele-specific amplification method principle, difference is that designed primer is 4, that is: 5 ' terminal sequence complementation of primers F 1 and equipotential X gene to be measured; 3 ' terminal sequence complementation of primer R2 and equipotential Y gene to be measured; Primer R1 is positioned at the middle and lower reaches of X gene sequence, and its 3 ' end is X; Primers F 2 is positioned at the middle and upper reaches of 5 ' gene order, and its 3 ' end is Y.X, Y represent polymorphic site respectively.When polymerase chain reaction (PCR) amplification, can amplify two oligonucleotide sequences that include mononucleotide polymorphism site simultaneously.Can identify target gene through a PCR.Though this method operation is simple, economical, owing to be to carry out in liquid phase, multiple parallel reaction limited amount is so be difficult to realize the high throughput testing of single nucleotide polymorphism.
Primer extension (Primer extension) is to rely on archaeal dna polymerase to differentiate class methods in nucleotide polymorphisms site, and its specificity is than the method height that is based upon the equipotential specific hybrid.These class methods have multiple title, as: micrometering preface (Minisequencing), mononucleotide primer extend (single nucleotide Primer extension, be abbreviated as SnuPE), the nucleotide synthetic (Primer-guided nucleotide incorpration) that instructs of primer, mix technology (template directed dye terminator incorpration is abbreviated as TDI) etc. based on template guided terminal fluorescence.Wherein, micrometering preface (Minisequencing) is the most commonly used.The reaction of micrometering preface at first amplifies the section of DNA that contains mononucleotide polymorphism site, carry out the reaction of micrometering preface then, add one and detect primer, its 3 ' terminal bases is adjacent in the polymorphism base, carry out the extension of a base in the presence of the ddNTP of archaeal dna polymerase and mark, this base of extension is exactly the polymorphism base.Because PCR primer meeting and the competition of extension primer produce a plurality of amplified fragments, and unnecessary dNTP can make extension extend a plurality of bases.So, must carry out purifying to the PCR product before the reaction of micrometering preface, to remove PCR primer and the dNTP that wherein contains.The detection of primer extension product has labelled with radioisotope method, luminous detection method, gel to be several different methods such as the fluorescence detection on basis and mass spectrometry, sex change high pressure lipuid chromatography (HPLC).But these methods are in various degree defective that has cost height, complex operation or deficiency all.As: the labelled with radioisotope method, the automaticity of luminous detection method is higher, and more convenient, but the cost of equipment needed thereby is higher; Gel is the loaded down with trivial details effort of fluorescence detection manufacturing process on basis.
Going up the method that single nucleotide polymorphism is analyzed based on allele specific nucleotide fragments (Allele specific oligonucleotide is abbreviated as ASO) hybridization basis, is the simplest detection method based on the hybridization principle.When it is hybridized according to short nucleotide probe and complementary purpose fragment, mate fully with two kinds of situations of mispairing are arranged under hybridization complex stability different, mononucleotide polymorphism site is detected.This method at first will design the nucleotide probe of one section weak point, is generally 15-20bp, comprising mononucleotide polymorphism site; When the hybridization of itself and sample DNA, because the difference of a base can cause Tm value decline 5-7.5 degree in 20bp, so, control hybridization conditions by strictness, just can identify whether there is single nucleotide polymorphism in the sample DNA.Shortcoming is that the severity of hybridization is wayward, can not distinguish mononucleotide polymorphism site more accurately.
In order to improve the severity of hybridization,, then need adopt the nucleotide probe and the sample DNA hybridization of modified, for example: utilize peptide nucleic acid (Peptide nucleic acids is abbreviated as PNA) probe better to distinguish mononucleotide polymorphism site.But because peptide nucleic acid is made the probe poorly soluble, so reaction is difficult for carrying out.And the length of probe should be 7 bases at least, just can guarantee at room temperature to hybridize preferably.To being rich in the probe of guanine rhodamine mark, also there is the high problem of background fluorescence (FP) signal etc.Be reported in insert artificially in the probe base mismatch (3 '-nitropyrrole), during with this probe hybridization that contains base mismatch, to cause the decline of Tm value be 2 times of conventional hybridization to the difference of a base between probe and the purpose fragment, can improve the specificity of hybridization.But this method needs strict control hybridization conditions, and the accuracy that detects is not high.
The TagMan technology is based on the hybridization principle, the PCR mode is carried out the method for outer-gene amplification routinely.This method is to add in the system of PCR at mononucleotide polymorphism site and flanking sequence designed probe, probe has only the difference of a base, correspond respectively to different allele, and use fluorescence labeling probe, probe end is by phosphorylation, to prevent that probe is extended this probe and can carries out specificity with target sequence and combine in amplification procedure.When probe is complete, because the existence of quenching group by FRET (fluorescence resonance energy transfer) (FRET) technical role, can make fluorescence labeling not fluoresce; When carrying out polymerase chain reaction (PCR) amplification, utilize TagMan archaeal dna polymerase 5 '--3 ' end 5 prime excision enzyme activity, probe 5 ' end fluorescence labeling is scaled off from probe, discharge fluorescence signal.Owing in the process of PCR, carry out, need not to separate or elution process, reduced the possibility of pollution of polymerase chain reaction, and the amplified production analysis is easy, quick, accurate, need not electrophoresis, the speed of experiment height.But, because designed probe only has the difference of a base.There is false positive in testing result, so the method for operating complexity detects the cost height, is unsuitable for applying.
Also there is following shortcoming in prior art:
1. the workload of Jian Ceing is big and the time is long.Adopt method for detecting single nucleotide polymorphism such as common PCR, fluorescent quantitative poly chain reaction, once can only detect, detect when being difficult to carry out a large amount of, multiple mononucleotide polymorphism site at one or more mononucleotide polymorphism sites.
2. fixing probe all is the special probes of species on the traditional biological chip, so the species that chip can detect fix, as if wanting to change or the interpolation detected object can only be made chip again.
Summary of the invention
The object of the present invention is to provide a kind of method of detecting mononucleotide polymorphism with biochip, it has solved and has been difficult to realize high throughput testing, the high technical matters of detection cost in the background technology.
Technical solution of the present invention is:
A kind of method of detecting mononucleotide polymorphism with biochip is characterized in that: the performing step of this method is as follows
1) sample of nucleic acid is increased
Get sample of nucleic acid, carry out amplified reaction, the nucleic acid fragment that minority is contained pleomorphism site increases the nucleic acid fragment that contains same pleomorphism site in a large number, obtains amplified production;
2) carry out the reaction of solution hybridization and ligase
(1) carry out solution hybridization: add difference probe and general probe, carry out solution hybridization with amplified production, must be through the amplified production of solution hybridization;
(2) carry out the ligase reaction: carry out the ligase reaction, the probe of coupling fully in the solution hybridization is connected with general probe, to detect the polymorphism of mononucleotide;
3) carry out solid-phase hybridization: with 5 ' end has probe and the biochip that label probe, 3 ' end has detection moiety and hybridizes, 25 ℃-75 ℃ of temperature, and time 0.5h-36h, detection moiety is assigned to the diverse location of biochip;
4) carrying out single nucleotide polymorphism detects: fluorescence kind, brightness and the distribution of detection moiety on the detection of biological chip diverse location get testing result.
Above-mentioned ligase reaction can be adopted the reaction of high temperature ligase, and performing step is as follows:
1) amplified production through solution hybridization is carried out purifying, remove enzyme unnecessary in the amplified production and primer;
2) carry out ligase reaction: adopt high temperature resistant ligase, in the PCR instrument, carry out sex change, connect cyclic process, mate probe fully and be connected, become one 5 ' hold to have label probe, 3 ' end and have the probe of detection moiety with general probe.Adopt high temperature resistant ligase, reaction velocity is fast, the joint efficiency height.
Above-mentioned purifying adopts PCR product purification kit, phenol chloroform extraction method, magnetic ball partition method or alkaline phosphatase and excision enzyme I facture etc. that amplified production is carried out purifying all can, simple to operate with the method that adopts PCR product purification kit purifying amplified production, and the purification efficiency height.
Above-mentioned ligase reaction can be adopted common ligase reaction, and performing step is as follows:
Adopt common ligase, under 0 ℃ of-25 ℃ of temperature, mate probe fully and be connected with general probe, become one 5 ' end has label probe, 3 ' end and has the probe of detection moiety.Adopt common ligase, amplified production does not need purifying, can directly connect.
Above-mentioned difference probe is to possess following condition:
1) base according to each pleomorphism site changes, and adopts two to four difference probes correspondingly;
2) 5 of every difference probe ' end all is the label probes that have the Different Alkali basic sequence, can be fixed on biochip on corresponding label complementary probe generation specific hybrid;
3) wherein a difference probe is to mate probe fully, its 3 ' terminal base can with polymorphic position dot blot in the amplified production;
4) all the other difference probes are not exclusively to mate probe, its 3 ' terminal base not with amplified production in the polymorphic position dot blot;
5) the sequence base portion complementation of the center section of every difference probe and amplified production.
Above-mentioned general probe is to possess following condition:
1) each pleomorphism site adopts a corresponding with it general probe;
2) general probe 5 ' end has phosphate group; First base of 5 ' end and first base complementrity behind the pleomorphism site;
3) general probe 3 ' end have can detected system identification detection moiety.
4) the sequence base portion complementation of general probe center section and amplified production.
Above-mentioned detection moiety can be fluorescein, rhodamine, green fluorescent protein, quantum dot, collaurum, magnetic ball or biotin.The preferred Cy3 of detection moiety, it is easy to operate, can directly detect, and highly sensitive.
Above-mentioned sample of nucleic acid is increased adopts PCR or constant-temperature amplification etc. all can.Constant-temperature amplification need not the cyclic process of temperature variation, and required instrument is simplified, and the reaction time is short.
Above-mentioned biochip can adopt modes such as in-situ synthesis, pin mark method or ink-jet method to make, and the solid phase carrier of described biochip can be microballoon, nylon membrane, cellulose acetate film or glass sheet etc.Can guarantee that the label complementary probe is efficiently fixed and the solid phase carrier on it all can.The biochip that adopts the pin mark method to make, the operation of biochip manufacturing process is simpler.Glass sheet is as solid phase carrier, and is simple to operate, label complementary probe fixed efficiency height, and cost is low.
The above-mentioned single nucleotide polymorphism of carrying out detect to adopt and directly to detect or indirect detection all can.
Above-mentioned solid phase hybridization is good with 60 ℃ of temperature, time 1h.Under this temperature not only the time of solid-phase hybridization short, and efficient height.
Invention has the following advantages:
1. hybridization efficiency height, the non-specific binding rate is low.
The present invention organically combines nucleic acid amplification, ligase reaction (LDR) and biochip, high specificity, and the hybridization efficiency height, crisscrossing, the non-specific binding rate is low.
2. accuracy rate height, false positive rate is low.
Adopt biochip test, the accuracy rate height, false positive rate is low.
3. low-cost, easy and simple to handle.
The present invention increases to the parent acid sample by the PCR, difference by single nucleotide in the DNA sample of nucleic acid of ligase reaction detection amplification back, by detecting hybridization signal with biochip hybridization, obtain testing result, when reducing cost, simplified operation steps.
4. can detect multiple mononucleotide polymorphism site.
The present invention is in the fixing a large amount of label complementary probe (antitag) of biochip surface, and therefore the mononucleotide polymorphism site that different label complementary probe is corresponding different, can detect thousands of kinds of different mononucleotide polymorphism sites simultaneously.
5. when detecting multiple mononucleotide polymorphism site, have high-throughout characteristics concurrently.
High flux can significantly reduce workload and the time that mononucleotide polymorphism site detects, and when multiple mononucleotide polymorphism site was detected, advantage was remarkable.For example, detect the mononucleotide polymorphism site of 20 kinds of different pathogenic microorganisms, approximately only need 16 hours, wherein 8 hours hybridization time need not staff's operation, has not only saved half the time, has also significantly reduced workload.If will detect thousands of kinds of mononucleotide polymorphism sites, advantage is more remarkable.
6. detection accuracy height.
The present invention adopts the ligase reaction technology to detect the sample of nucleic acid row labels of going forward side by side, and can fully guarantee the accuracy of net result.The ligase reaction technology adopts two class probes to detect a site, a class probe joint detection group wherein, another kind of connection label probe (tag).When the amplified production hybridization of two probes and sample of nucleic acid, when the amplified production of and if only if two probes and sample of nucleic acid mates fully, under the effect of ligase, 5 ' phosphate group between two probes is connected with 3 ' oh group, finally makes to detect detection moiety on the product band.Because the ligase reaction technology used two probes, can be compared to " Lock with double safety guard ", be that two probes must be all mate fully with the amplified production of sample of nucleic acid in the ligase reaction, ensuing coupled reaction could normally be carried out.That is, determine a nucleotide sequence by two probes, detect a kind of pleomorphism site, therefore, accuracy improves greatly, has avoided false positive results.
The hybridization reaction that takes place on the biochip carries out between label probe and label complementary probe.Crisscrossing (cross hybrization) is the bottleneck problem in the biochip hybridization process.Because biological genomic restriction own can't design the identical probe of specificity height and hybridization efficiency, but biochip of the present invention can address this problem.Because the label complementary probe of final hybridization is a kind of random series, screen by software, only and with the label probe of its base complementrity hybridize, and do not hybridize with other any probes.Because the sequence of these label complementary probe all is that high flux screening comes out, can guarantee that in design process it has very approaching fusing point (Tm), promptly can adopt same hybridization temperature, therefore guarantee probe under unified temperature, all have the same hybridization efficiency.Be to improve specificity, probe all has higher annealing temperature usually when design, can reach 65 ℃, hybridizes in higher temperature guaranteeing, can reduce possibility of non-specific hybridization greatly, further improves the accuracy of detection.
7. detection sensitivity height.
The present invention adopts the hybridizing method of solution hybridization and solid-phase hybridization combination, can fully ensure the accuracy of hybridization.The first step at hybridization reaction is the amplified production hybridization that has difference probe and the general probe and the sample of nucleic acid of label probe, is reflected in the liquid phase and carries out.The advantage of solution hybridization be probe can with the amplified production maximum space of sample of nucleic acid contact, sterically hindered reducing, the ability of the amplified production of probe capture nucleic acid sample improves greatly, so hybridization efficiency improves.That is to say that the probe of equal number can capture the amplified production of more sample of nucleic acid, sensitivity is improved.
Solution hybridization reactant liquor and biochip are hybridized, essence is that the label complementary probe fixing on the biochip and the label probe of solution hybridization reactant liquor are hybridized, these two sections probes can be avoided and other probe mispairing, the more traditional biochip probe of sensitivity of hybridization is high a lot, add that its annealing temperature is higher, the efficient of hybridization improves greatly, and therefore, detection sensitivity of the present invention also is further enhanced.
8. detection speed is fast.
Because the melting point values of fixing label complementary probe is higher on the biochip of the present invention, and the hybridization specificity between label complementary probe and the label probe is very high,, reduced detection time greatly so the time of solid-phase hybridization only needs 1 hour.
9. the opening that has system.
The probe that is fixed among the present invention on the biochip is and the irrelevant label complementary probe of detected object, the probe that the change detected object only need be changed in the solution hybridization system gets final product, the probe that is fixed on the biochip then need not to change, so the biochip of making can be used for the detection of different single nucleotide polymorphism.
10. cost is low.
The cost of biochip maximum is from probe, and the biochip among the present invention can be saved the cost of probe because the characteristic of itself has avoided repeating a coremaking sheet.
Generally, probe is long more during hybridization, and the accuracy of hybridization, confidence level are high more.The present invention is under the very high situation of the efficient of guaranteeing label probe and label complementary probe specific hybrid, and probe length fixing on the chip approximately is 20 bases, has saved the synthetic cost of probe.
In addition, because the high flux property of biochip itself can be used for the detection of thousands of kinds of different mononucleotide polymorphism sites simultaneously, greatly reduce the detection cost.
11. characteristics such as that the present invention has is easy, quick, sensitive, specificity height have wide range of applications.The present invention can detect the trace dna sample, can in the polymorphic sexual type of thousands of height flat line modes, obtain genotype, also can be applicable to the losing and correlative study or the like of diagnosis, medical diagnosis on disease, searching Disease-causing gene seat, quantitative trait locus analysis, cancer diagnosis genetic heterozygosis of pathogenic microorganism.
Description of drawings
Fig. 1 is a detection mechanism synoptic diagram of the present invention;
Fig. 2 is the biochip hybridization collection of illustrative plates of experimental result of the present invention.
Embodiment
Nucleic acid amplification technologies is the technology that a kind of economic fast synthetic a large amount of dna fragmentations copy.Be usually used in the pre-natal diagnosis of hereditary disease, detection of oncogene and diagnosis, the detection of infective pathogen body, dna fingerprint, individual identification, parent are differentiated and forensic, the animal and plant quarantine, quick diagnosis detects, gene splicing, order-checking equimolecular field of biology.
The ligase reaction is the technology that two nucleic acid fragments are coupled together, and is usually used in molecular cloning equimolecular field of biology.
Biochip has the susceptibility and the specificity of height, it can monitor several situations of transcribing to several thousand mRNA copy in the cell, thus aspect gene expression analysis as: analyzing gene is expressed space-time characteristic, gene differential expression detects, finds that all there is a lot of application aspects such as new gene, extensive dna sequencing.And, also be widely used in genotype, gene mutation and extensive polymorphism analysis just because of the high flux property of biochip and sensitivity can analyze the faint difference between a plurality of similar genes.Biochip also is that the strong tool of medical diagnosis on disease and treatment detects as: the location of hereditary disease related gene, the diagnosis of infectious diseases, antibody-resistant bacterium and susceptibility in addition.
The present invention organically combines nucleic acid amplification technologies, ligase reaction (ligase detection reaction is abbreviated as LDR) and biochip, is mainly used in the pleomorphism site that detects in the nucleotide sequence.Can detect the pleomorphism site of a plurality of genes in the pleomorphism site of individual gene in the different samples or a kind of sample simultaneously.
Referring to Fig. 1, it is as follows that the present invention detects step:
1. sample of nucleic acid is increased.
For being contained the nucleic acid fragment of mononucleotide polymorphism site in a large number, need increase to sample of nucleic acid.The nucleic acid fragment that employing can contain minority pleomorphism site increases the method for the nucleic acid fragment that contains same pleomorphism site in a large number all can.For example, and the PCR (Polymerase Chain Reaction, PCR), constant-temperature amplification etc.The PCR is that sample of nucleic acid is carried out the PCR, obtains amplified production.The topmost characteristics of constant-temperature amplification are to react all to carry out under same temperature.As: (Nucleic acidsequence-based amplification NASBA), can pass through reverse transcriptase, RNA enzyme H, three kinds of enzyme synergies of RNA polymerase, synthesizing single-stranded DNA of while, double-stranded DNA and single stranded RNA in the amplification of nucleotide sequence.
Get sample of nucleic acid, carry out amplified reaction, the nucleic acid fragment that minority is contained pleomorphism site increases the nucleic acid fragment that contains pleomorphism site in a large number, obtains amplified production.Sample of nucleic acid increased to adopt PCR or constant-temperature amplification etc.Constant-temperature amplification need not the cyclic process of temperature variation, and required instrument is simplified, and the reaction time is short.
2. solution hybridization and ligase reaction.
For introducing the detection moiety that to discern mononucleotide polymorphism site, be convenient to detection system and detect.Specific embodiments is as follows:
1) solution hybridization: add difference probe (discriminating oligo), general probe (common probe) and amplified production and carry out solution hybridization, make the amplified production sex change, must be through the amplified production of solution hybridization.
The difference probe that adds in the solution hybridization must satisfy following condition:
(1) base according to each pleomorphism site changes, and adopts two to four difference probes correspondingly.
(2) 5 of every difference probe ' end all is the label probes that have the Different Alkali basic sequence, can be fixed on biochip on corresponding label complementary probe generation specific hybrid.
(3) wherein a difference probe is to mate probe (perfect match probe) fully, its 3 ' terminal base can with polymorphic position dot blot in the amplified production;
(4) all the other difference probes are not exclusively to mate probe (mismatch probe), its 3 ' terminal base not with amplified production in the polymorphic position dot blot;
(5) the sequence base portion complementation of the center section of every difference probe and amplified production.
The general probe that adds in the solution hybridization must possess following condition:
(1) each pleomorphism site adopts a corresponding with it general probe
(2) general probe 5 ' end has phosphate group; First base of 5 ' end and first base complementrity behind the pleomorphism site.
(3) general probe 3 ' end have can detected system identification detection moiety.Detection moiety can be a fluorescein, rhodamine, green fluorescent protein, magnetic ball, biotin and any material that can discern with testing laboratory's detection system.Detection moiety is selected Cy3 among the present invention, because easy to operate, can directly detect, and highly sensitive.
(4) the sequence base portion complementation of general probe center section and amplified production.
2) ligase reaction: under the effect of ligase, the probe of coupling fully in the solution hybridization is connected with general probe, to detect the polymorphism of mononucleotide.In the ligase reaction,, can be divided into reaction of high temperature ligase and the reaction of common ligase because of the difference of ligase character.Amplified production through solution hybridization is carried out purifying, can remove enzyme unnecessary in the amplified production and primer.
Adopt high temperature resistant ligase, as: resistant to elevated temperatures dna ligase (Taq DNA ligase), in the PCR instrument, carry out sex change, connect cyclic process, mate probe fully and be connected, become one 5 ' hold to have label probe, 3 ' end and have the probe of detection moiety with general probe.Adopt high temperature resistant ligase, reaction velocity is fast, the joint efficiency height, but amplified production must be earlier through purifying.
Purification process: amplified production is carried out purifying with PCR product purification kit, phenol chloroform extraction method, magnetic ball partition method or alkaline phosphatase and excision enzyme I facture etc.Simple to operate with the method that adopts PCR product purification kit purifying amplified production, and the purification efficiency height.
Adopt common ligase,, under 0 ℃ of-25 ℃ of temperature, mate probe fully and be connected with general probe as the T4 ligase, become one 5 ' end has label probe, 3 ' end and has the probe of detection moiety.Adopt common ligase, amplified production does not need purifying, can directly connect, but reaction velocity is slow, and joint efficiency is low.
3. solid-phase hybridization.
Two kinds of different nucleic acid molecules are by the base complementrity combination, and base is passed through hydrogen bonded.Hybridize and actually occur in 5 ' terminal label probe and being fixed between the label complementary probe on the biochip.5 ' end is had probe and the biochip hybridization that label probe, 3 ' end has detection moiety, and hybridization temperature is 25 ℃-75 ℃, and hybridization time is 0.5h-36h, and detection moiety is assigned to the diverse location of biochip.Detection moiety can detected system identification.When hybridization temperature was 60 ℃, hybridization time was short, only is 1h; And hybridization efficiency height.
The biochip that uses in solid-phase hybridization is divided into according to production method and the different of solid phase carrier: that modes such as in-situ synthesis, pin mark method, ink-jet method are made, can guarantee the label complementary probe efficiently fixing with its on biochip.The biochip that adopts the pin mark method to make, biochip manufacturing process is fairly simple.The solid phase carrier of energy such as microballoon, nylon membrane, cellulose acetate film, glass sheet held stationary label complementary probe.Glass sheet is as solid phase carrier, and is simple to operate, label complementary probe fixed efficiency height, and cost is low.
4. detection single nucleotide polymorphism.
Results of hybridization is carried out check and analysis, and fluorescence kind, brightness and distribution according to detection moiety on the biochip diverse location can obtain testing result.Detection method adopts directly detection or indirect detection all can.
Directly detect: the direct detected recognition of devices of detection moiety is Cy3, Cy5 and various fluorophor, quantum dot etc. as: detection moiety.Adopt the method that directly detects, simple to operate, highly sensitive.
Indirect detection: method that just can detected recognition of devices after detection moiety must further react with other materials.As: detection moiety is a biotin, magnetic ball etc.
Experiment: identify eight kinds of food-borne pathogens such as staphylococcus aureus, enterohemorrhagic Escherichia coli 0157:H7, Bacterium enteritidis, small intestine colon Yersinia ruckeri, monokaryon hyperplasia Lee Salmonella, Shigella flexneri, bacillus cereus, A type perfringens bacillus simultaneously.
Operation steps:
1. get the sample of nucleic acid of staphylococcus aureus, enterohemorrhagic Escherichia coli 0157:H7, Bacterium enteritidis, small intestine colon Yersinia ruckeri, monokaryon hyperplasia Lee Salmonella, Shigella flexneri, bacillus cereus, eight kinds of bacterium of A type perfringens bacillus, i.e. genomic DNA.
2. with the PCR sample of nucleic acid is increased.
3. carry out the reaction of solution hybridization and ligase.
4. carry out solid-phase hybridization
5. the product of the Axon Genepix 4000B cake core scanner of producing with Axon company after to solid-phase hybridization detects, laser " PMT voltage " at the 532nm passage is made as 600, " Power " all is made as 100% pair of biochip and scans, obtain the biochip hybridization collection of illustrative plates, referring to Fig. 2.
6. the hybridization collection of illustrative plates is carried out analytical proof: the minimum amplified production that detects 20ng of this experiment, minimum can detected total plate count be 110cfu/mL.
Sample of nucleic acid of the present invention, its nucleic acid or nucleic acid molecules refer to deoxidation nuclear (sugar nuclear) thuja acid or nuclear (sugar is examined) the thuja acid polymer of strand or double chain form existence, as do not have other restrictions, also comprise the material that similarity is arranged with natural acid.
1) nucleic acid that can be used for this test comprises interested pleomorphism site of researcher and the nucleic acid from coming according to pleomorphism site.Here the nucleic acid that comes according to pleomorphism site refers to the genomic DNA that finally is used for masterplate, wherein includes pleomorphism site.
(1) DNA that comes from genome amplification, the RNA that comes from DNA amplification transcribes and the mRNA that comes from genomic DNA, or from the mRNA reverse transcription and the cDNA that comes etc. are to come according to pleomorphism site.
(2) when not specifying, the sample of nucleic acid that can be applied to this test should comprise from the next DNA of genome amplification, from the next RNA of DNA amplification, transcribe and next RNA from genomic DNA, the cDNA that comes from the mRNA reverse transcription, from cDNA next cDNA, the DNA that from gene, amplifies, the RNA that from DNA amplification, transcribes and similar sample.
(3) if sample is not a dna sample, before amplification, at first need to be translated into double-stranded DNA, can with reverse transcriptase and (or) archaeal dna polymerase.
2) sample of nucleic acid can be from single organism, as: the people, animal, plant or microorganism also can be from multiple microorganisms, and at this moment testing result can disclose the frequency that sample of nucleic acid allele occurs.
3) sample of nucleic acid should be the uniform mix of cell, tissue or other biological sample.
(1) to be fit to total DNA be masterplate to biological specimen, also can be total RNA sometimes, and total RNA comprises and has the ripe mRNA of Poly-A and the mRNA that is synthesizing.
(2) biological sample refers to from any organism and the biological tissue, body fluid or the cell that come.
Usually these samples all are clinical samples, and clinical sample can provide the abundant information of various allele and its relevant disease.
This invention specifically can detect gene mutation and differentiate mutant phenotype.The value so this invention has a wide range of applications in clinical diagnosis and clinical research.
Clinical sample comprises body fluid, blood plasma, haemocyte (as leucocyte), tissue, marrow or the like.
Biological specimen also comprises other tissues, as sample frozen or that soak with formalin solution.
In addition, cell culture also is the biological specimen of a quasi-representative, and it can be a clinical sample, initial cell culture, and subculture, any organism clone can be as the source of DNA and RNA sample.
The explanation of main agents of the present invention:
Taq archaeal dna polymerase: the product that can adopt precious bioengineering (Dalian) company limited.
Taq dna ligase: the product that can adopt U.S. NEB company to produce.
The Tris-HCl damping fluid: but the laboratory preparation.
Picodna triphosphoric acid potpourri dNTPs: the product that can adopt American I nvitrogen company to produce.
Specific PCR primer: can adopt the synthetic product of Beijing three rich polygala root biotechnology Ltds.
Difference probe: can adopt the synthetic product of Beijing three rich polygala root biotechnology Ltds.
General probe: can adopt the synthetic product of Beijing three rich polygala root biotechnology Ltds.
Tag probe: can adopt the synthetic product of Beijing three rich polygala root biotechnology Ltds.
Antitag probe: can adopt the synthetic product of Beijing three rich polygala root biotechnology Ltds.
PCR product purification kit: the product that can adopt U.S. QIAGEN company to produce.
Claims (10)
1. the method for a detecting mononucleotide polymorphism with biochip, it is characterized in that: this method may further comprise the steps
1) sample of nucleic acid is increased
Get sample of nucleic acid, carry out amplified reaction, the nucleic acid fragment that minority is contained pleomorphism site increases the nucleic acid fragment that contains same pleomorphism site in a large number, obtains amplified production;
2) carry out the reaction of solution hybridization and ligase
(1) carry out solution hybridization: add difference probe and general probe, carry out solution hybridization with amplified production, must be through the amplified production of solution hybridization;
(2) carry out the ligase reaction: carry out the ligase reaction, the probe of coupling fully in the solution hybridization is connected with general probe;
3) carry out solid-phase hybridization: with 5 ' end has probe and the biochip that label probe, 3 ' end has detection moiety and hybridizes, 25 ℃-75 ℃ of temperature, and time 0.5h-36h, detection moiety is assigned to the diverse location of biochip;
4) carrying out single nucleotide polymorphism detects: fluorescence kind, brightness and the distribution of detection moiety on the detection of biological chip diverse location get testing result.
2. the method for detecting mononucleotide polymorphism with biochip according to claim 1 is characterized in that: described ligase reaction is reacted for the high temperature ligase, and performing step is as follows:
1) amplified production through solution hybridization is carried out purifying;
2) carry out ligase reaction: adopt high temperature resistant ligase, in the PCR instrument, carry out sex change, connect cyclic process, mate probe fully and be connected, become one 5 ' hold to have label probe, 3 ' end and have the probe of detection moiety with general probe.
3. the method for detecting mononucleotide polymorphism with biochip according to claim 2, it is characterized in that: described purifying is with PCR product purification kit, phenol chloroform extraction method, magnetic ball partition method or alkaline phosphatase and excision enzyme I facture amplified production to be carried out purifying.
4. the method for detecting mononucleotide polymorphism with biochip according to claim 1 is characterized in that: described ligase reaction is common ligase reaction, and performing step is as follows:
Adopt common ligase, under 0 ℃ of-25 ℃ of temperature, become one 5 ' end has label probe, 3 ' end and has the probe of detection moiety.
5. according to the method for claim 1 or 2 or 4 described detecting mononucleotide polymorphism with biochip, it is characterized in that: described difference probe possesses following condition:
1) base according to each pleomorphism site changes, and adopts two to four difference probes correspondingly;
2) 5 of every difference probe ' end all is the label probes that have the Different Alkali basic sequence, can be fixed on biochip on corresponding label complementary probe generation specific hybrid;
3) wherein a difference probe is to mate probe fully, its 3 ' terminal base can with polymorphic position dot blot in the amplified production;
4) all the other difference probes are not exclusively to mate probe, its 3 ' terminal base not with amplified production in the polymorphic position dot blot;
5) the sequence base portion complementation of the center section of every difference probe and amplified production;
Described general probe possesses following condition:
1) each pleomorphism site adopts a corresponding with it general probe;
2) general probe 5 ' end has phosphate group, first base of 5 ' end and first base complementrity behind the pleomorphism site;
3) general probe 3 ' end have can detected system identification detection moiety;
4) the sequence base portion complementation of general probe center section and amplified production.
6. the method for detecting mononucleotide polymorphism with biochip according to claim 5, it is characterized in that: described detection moiety is fluorescein, rhodamine, green fluorescent protein, quantum dot, collaurum, magnetic ball or biotin.
7. the method for detecting mononucleotide polymorphism with biochip according to claim 5 is characterized in that: described sample of nucleic acid is increased is to adopt PCR or constant-temperature amplification.
8. the method for detecting mononucleotide polymorphism with biochip according to claim 7, it is characterized in that: described biochip adopts in-situ synthesis, pin mark method or ink-jet method mode to make, and the solid phase carrier of described biochip is microballoon, nylon membrane, cellulose acetate film or glass sheet.
9. the method for detecting mononucleotide polymorphism with biochip according to claim 8 is characterized in that: described to carry out that single nucleotide polymorphism detects be to adopt directly to detect or indirect detection.
10. the method for detecting mononucleotide polymorphism with biochip according to claim 9, it is characterized in that: described solid-phase hybridization temperature is 60 ℃, the solid-phase hybridization time is 1h.
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