CN106754343B

CN106754343B - DNA sequencing apparatus and system based on pyrosequencing

Info

Publication number: CN106754343B
Application number: CN201710061926.5A
Authority: CN
Inventors: 刘丹; 孙悦
Original assignee: Wuhan First Biological Technology Co Ltd
Current assignee: Wuhan Feisite Biotechnology Co ltd
Priority date: 2017-01-12
Filing date: 2017-01-27
Publication date: 2017-10-31
Anticipated expiration: 2037-01-27
Also published as: CN106754343A; CN107299054B; CN107299054A; CN106591107A; CN107312710B; CN106754313A; CN106754292B; CN106754292A; CN107312710A; CN106591107B

Abstract

The invention provides a kind of DNA sequencing system based on pyrosequencing, including sample area, reaction zone, detection zone and control zone；The sample area includes rotatable separator disk, the separator disk includes at least one DNA Disengagement zone, the Disengagement zone includes the internal hollow Filter column for being provided with filter membrane, is connected with the single-stranded separation after the membrane filtration of DNA of the single-stranded and not connected affine connectors of the DNA of affine connector；The reaction zone includes sample-adding portion and sample cell, and the sample-adding portion includes dNTP reagent troughs, sample-adding punch block and the multiple sample needles being mounted thereon；The sample-adding punch block is located on sample cell；The sample cell is provided with multiple grooves position；The detection zone includes bioluminescent detection device, and the groove position is connected with bioluminescent detection device；The sample area, reaction zone and detection zone are monitored by control zone, controlled.The DNA sequencing apparatus and system based on pyrosequencing of the present invention has the advantages that easy to operate, quick detection.

Description

DNA sequencing apparatus and system based on pyrosequencing

Technical field

The present invention relates to a kind of DNA sequencing device based on pyrosequencing, it is specifically related to a kind of based on pyrophosphoric acid survey The DNA sequencing apparatus and system based on pyrosequencing of sequence, belongs to field of gene detection.

Background technology

First, pyrosequencing overview

Pyrosequencing techniques (Pyrosequencing) be grew up in 1987, based in DNA building-up processes The sequencing technologies of pyrophosphoric acid (PPi) detection of release, pyrosequencing is reacted under a series of catalytic action of enzymes, its process It is middle to produce the visible ray proportional to the aggregate number of deoxynucleotide triphosphates (dNTP), by can reach to visible detection Determine the purpose of DNA sequence dna.Pyrosequencing has two kinds of implementation methods：Liquid phase pyrosequencing method (Liquid Phase ) and solid phase pyrosequencing method (Solid Phase Pyrosequencing) Pyrosequencing.

Liquid phase pyrosequencing is to cascade chemiluminescence reaction by the enzyme in the same reaction system of 4 kinds of enzymatics, and it is former Reason is：After primer is annealed with template DNA, in archaeal dna polymerase (DNA polymerase), ATP sulfurylases (ATP s μ Lfurylase), under luciferase (1uciferase) and apyrase (Apyrase) synergy, by primer Each upper dNTP of DNA polymerization and the release coupling of fluorescence signal are got up, and by detecting release and the intensity of fluorescence, reach reality When determine purpose (horse Yongping etc., pyrosequencing techniques and its external in application [J] of biology field of DNA sequence dna Medical Molecular Biology fascicle 2003,25 (2):115-117).

The reaction system of liquid phase pyrosequencing is made up of reaction substrate, single-stranded, specific sequencing primer and enzyme to be measured, instead It is 5 '-phosphosulfate (APS) and fluorescein (1uciferin) to answer substrate.Liquid phase pyrosequencing course of reaction is one to anti- Answer and add the process that 4 kinds of dNTP participate in reaction in system in turn, only a kind of dNTP of often wheel reaction is participated in.If the dNTP added Next base pairing that can just be with DNA profiling, then its 3 ' end that sequencing primer is added in the presence of archaeal dna polymerase End, while discharging the pyrophosphoric acid (PPi) of a molecule；In the presence of ATP sulfurylases, PPi the and APS combination shapes of generation Into ATP；In the presence of luciferase, the ATP of generation is again and fluorescein combines to form oxyluciferin, while producing visible Light.If this dNTP added can just with the individual identical Mismatchings of continuous n below DNA profiling, according to reaction equation Understand, n times when the visual intensity discharged should be only 1 Mismatching, namely the luminous intensity discharged in course of reaction Relation proportional to the base number matched.If the dNTP and next base of DNA profiling that add are mismatched, above-mentioned Reaction will not occur, the also release without visible ray.The dNTP and ATP of reaction are not participated in nucleolytic enzyme Apyrase's Effect is lower to degrade.

The visible ray that often wheel reaction is discharged is converted by Weak light detection device, then is treated as data signal, through PC Software processing can obtain a special detection peak, and the height of peak value should be with the proportional relation of base number matched.

After last round of reaction terminates, another dNTP is added, repeats above-mentioned reaction.Finally can be according to the light of acquisition Intensity peak figure is the DNA sequence dna information to be measured that can be read.

It should be noted that：DATP catabolite deoxidation Guanosine 5'-Monophosphate (dAMP) is the inhibitor of luciferase, with The progress of reaction, its concentration meeting more and more higher can hinder proceeding for pyrosequencing chemiluminescence reaction.This is also burnt Main cause (Shendure J, the et a1.Advanced of phosphoric acid PCR sequencing PCR sequencing length shorter (being usually 20bp~30bp) sequencing technologies:Methods and goals, Nat.Rev Genet., 2004,5 (5):335-44).

Solid phase pyrosequencing is by the chemiluminescence reaction of 3 kinds of enzymatics, compared with liquid phase pyrosequencing, without three AMP bisphosphatase is participated in.Solid phase pyrosequencing course of reaction is as follows：The DNA profiling for combining primer is fixed in branch Support on thing and holding position is constant during the course of the reaction；Add after a kind of dNTP, in archaeal dna polymerase, ATP sulfurylases and fluorescence Reacted under plain enzyme synergy, except not having degradation reaction, other reactions are identical with liquid phase pyrosequencing； There is a rinsing step (washing step) to wash away upper wheel reaction residue completely before a kind of dNTP under addition, do not have The accumulation of inhibition product.

Under normal circumstances, the pyrosequencing method described in people refers to liquid phase pyrosequencing method, because its four enzyme is anti- Answer system that pyrosequencing is easily realized in single tube.

Ronaghi etc. substitutes signal to noise ratio (the Ronaghi M.et that dATP improves pyrosequencing using dATP α S a1.Real-time DNA sequencing using detection of PPi release；Anal.Biochem, 1996, 242(1):84-89).Because dATP α S can more effectively be utilized by archaeal dna polymerase than dATP, it is more beneficial for reading rich in T Region.DATP α S are two kinds of isomers Sp-dATP α S and Rp-dATP α S mixtures, and polymerase can only utilize Sp-dATP α S. In order to obtain optimum response efficiency, it is necessary to the Sp-dATP α S of optium concentration are kept in reaction system, at the same time Rp-dATP α S concentration is also in increase.DATP α S can still produce the mortifier of luciferase after being degraded by Apyrase, thus dATP α S's plus Enter not to be improved reading Process capabi l i ty 32.

Gharizadeh et al. is improved this, and they only add pure Sp-dATP α S in the reaction, and are added without Useless Rp-dATP α S, improve the efficiency of reaction, greatly reduce the concentration for suppressing product so that luciferase can be tieed up The activity of long period is held, making the sequencing length of pyrosequencing method increases to 50bp~100bp, and the increase of sequencing length also makes Obtaining pyrosequencing techniques has many new application (Gharizadeh B.et al., Long-read pyrosequencing using pure 2’-deoxyadenosine-5’-0’-(1-thiotriphosphate)Sp-isomer；Anal Binchem, 2002.301:82-90).

The 12nd gene order-checking and analysis meeting (12th International Genome held in 2000 Sequencing and Analysis Conference) on, Ronaghi et al. proposes a kind of remove and suppresses product, reduces The method of dilution effect, 200bp is increased to by sequencing length.

Compared with sanger dideoxy chain-termination PCR sequencing PCRs, pyrosequencing method has quick, accurate, economic, inspection in real time The characteristics of survey；It does not need gel electrophoresis, it is not required that mark and the dyeing of any special shape are carried out to DNA sample, is had Very high repeatability；The automation of the concurrency and height of height can be realized.

2nd, the application progress of pyrosequencing techniques

1 application in SNP research

SNP (Single Nucleotide Polymorphisms, SNPs) is occurred in recent years Three generations's genetic marker, it refers to has two kinds of different bases in genome on specific nucleotide position, wherein minimum one kind It is not less than 1% in the frequency of colony.SNPs is most commonly seen genetic polymorphism type in biological genome, and it can be any one A series of marks are provided close to or within individual gene to be studied；And the polymorphism in exactly this genome, i.e. genome sequence The difference of row constitutes Different Individual and neurological susceptibility of the colony to disease, the science of heredity base to medicine and envirment factor differential responses Plinth.

SNP research mainly includes two aspects：One is the structure of snp database, mainly finds that particular types are biological All or part of SNP of genome.Two be SNP functional studies, and it is the first step of SNP researchs to find SNP, and SNP functions are ground Study carefully the purpose for being only SNP researchs.Sanger sequencing technologies have turned into extensive, accurate, the quick mainstream technology for finding SNP.And Sequence verification analysis and frequency analysis are carried out to existing SNP in database, the pyrosequencing of short sequence and checking is good at Technology is good selection, carries out SNP researchs using pyrosequencing techniques, can more save the time and reduce consumption.

Nordfors etc. is respectively adopted Taqman fluorescent quantitations and pyrosequencing techniques and entered to being up to 1022 samples Row SNP Genotypings study, obtain identical result, this control experiment show pyrosequencing techniques be carry out high flux, The SNPs of large sample efficient, high accuracy method.Wasson etc. carries out DNA ponds (DNA using pyrosequencing techniques Pools SNP gene frequencies analysis).Rickert etc. carries out gene using pyrosequencing techniques to 4 times of body potatos Type research, in 94 pleomorphism site detections, has 76 allele sites to be differentiated with pyrosequencing techniques, effectively Rate is up to 81%.Jiang Siwen etc. using pyrosequencing techniques differentiate the work of porcine mtdna cytochrome b gene haplotype Make.Yuan Jianlin etc. carries out HLA-DRB genotyping researchs using pyrosequencing techniques, and experiment shows, by pyrosequencing As a result with can accurately carry out Genotyping after the Gene sequence comparison of HLA databases, this method can be applied to clinical organ transplant Donor/acceptor examination.

2 application in pathogenic microorganism Rapid identification

Jonasson etc. detects pathogen 16S rRNA genes with pyrosequencing techniques, and Rapid identification goes out in clinical samples Antibiotic resistance bacterium.Monstein etc. successfully detects the V1 of helicobacter pylori 16S rRNA genetically labile with pyrosequencing techniques With V3 region sequences, it was demonstrated that the technology can meet the Rapid identification and parting to analysis of clinic pathogenic microorganism sample.Unnerstad etc. is utilized should Technology has carried out parting to the Listeria Monocytogenes of 106 plants of different serotypes, is existed using pyrosequencing techniques Substantial amounts of sample sequencing, its concurrency and high efficiency highly significant are completed in short time.Gharizadeh etc. is with this technology to 67 Individual HPV sample has carried out identification and parting, it was demonstrated that the technology is also very suitable for the extensive mirror of the pathogen such as HPV Fixed, parting and the research of mutation.Cheng Shaohui etc. extracts viral RNA from the Vero-6 cells of infection people SARS virus, using Jiao Phosphoric acid sequencing technologies multiple base mutation sites are sequenced and frequency of mutation analysis.Mutation is likely to occur by the way that sequencing analysis are multiple Site, it is determined that the virus be Beijing epidemic strain.

3 application in Study of Etiology

Kittles etc. utilizes pyrosequencing techniques, to Nigerian, European descendants American and African American three The CYPl7 gene pleiomorphisms of individual different population are analyzed, and study the CYPl7 gene pleiomorphisms and prostate of African American Relation and clinical manifestation between cancer.Result of study shows that sequence compares sequence for the African American of CC CYPl7 genotype The probability for suffering from prostate cancer for TT CYPl7 genotype African Americans wants height, it was demonstrated that the base in this kind of crowd is C's CYPl7 gene pleiomorphisms and the incidence of disease of prostate cancer are in close relations, are people at highest risk.Numerous clues show, positioned at chromosome There is important relation in 22q11 COMT genes, and the research work of scientist could not be proposed always with schizoid morbidity Strong evidence；Shifman etc. proposes a kind of effective method, and they utilize pyrosequencing techniques, to the moral of large sample It is that Jew crowd carries out related single nucleotide polymorphism analysis, it was demonstrated that exist between schizoid generation and CMOT genes The association of height.This method also can be suitably used for the genetic analysis research of other diseases.

4 application in forensic identification

With Sanger PCR sequencing PCRs to mitochondrial DNA (mtDNA) analysis of variance, it is impossible to realize to by containing pollutant, multiple The precise quantification analysis of the mtDNA mixtures of the compositions such as individual DNA, and Andreasson etc. proposes one kind and mixed for mtDNA The novel quantization method based on pyrosequencing techniques of analysis is closed, can be easily fast from court's exhibit mixing sample Speed, detect main and secondary mtDNA compositions exactly.Balitzki-Korte is using pyrosequencing techniques to line grain Body 12SrRNA genes carry out sequencing analysis, and the detection that length is 20bp is carried out on the gene segment of 149bp length, passes through ginseng Examine database sequence, it becomes possible to be able adequately determines the biology origin of object, such as, it can determine that one piece of skin histology is actually With missing crew or animal.

3rd, pyrophosphoric acid analytical equipment and development

The application of pyrosequencing techniques depends on the research and development of pyrophosphoric acid analytical equipment.No matter which kind of pyrophosphoric acid point Analysis apparatus, its primary structure should all include two parts：Reactor part and faint light detecting portion.Reactor provide react into Capable place, faint light detecting portion is responsible for the visible ray that detection reaction is sent.In research of the people to pyrosequencing techniques In application process, the reactor of design and use can be largely classified into 3 classes：Micro plate reactor, micro-fluidic chip reactor With micro-array chip reactor.

And commercialized pyrosequencing instrument has emerged abroad, but the report that domestic pertinent instruments are studied in itself is very It is few, more come out without corresponding product.One Typical Representative of external product is the PSQ96 of Pyrosequencing AB companies, it It is the product that the said firm releases for 2001, system can carry out the independent sequencing of 96 tunnels or 384 road DNA samples simultaneously, work as survey Accuracy and reliability reached 99%, with high pass at 45 minutes 1 hour the general time used during the of length no more than 300bp of sequence Amount, quick, economic advantage.PSQ96 systems are had been widely used among basic medical research and clinical molecular diagnosis.

It is the Genome that 454Life Science companies of the U.S. release for 2005 that external instrument, which studies another representative, Sequencer20(GS20).It has moved towards the miniaturization direction of more high technology content, i.e., using MEMS technology by micro-filtration chamber The reaction environment reacted as pyrosequencing, the reaction array of surprising numbers up to a million is integrated into 7cm × 8cm area It is interior, and enable each reaction cabin independently while carrying out sequencing cascade reaction, high sensitivity and the CCD energy of resolution ratio that instrument has The fluorescent signals that each single reaction cabin is produced enough are captured, the sequence information of each specimen dna can be finally obtained.GS20 Only need 4.5 hours that high density sequencing reaction can be achieved, the sequence information of each sample is obtained by parallel computation.Advantage is The consumption of reaction reagent can be saved, reduction sequencing cost provides possibility for genome large scale sequencing.

Instrument research domestic at present is at the early-stage, and the road of production domesticization is very long, the problem of in face of every aspect, in balance After various hardware conditions required for sequencing system, the problem of finding primary face is the development of micro sample-adding subsystem with challenge Problem.

4th, in pyrosequencing sample adding system importance

Pyrosequencing techniques and products thereof are big flux, low cost, in good time, quickly and intuitively progress mononucleotide is more State property is studied and clinical examination provides ideal technical operation platform, is the genome times afterwards comprehensively to carry out gene sequencing The powerful of research.Pyrosequencing techniques are just received and used by increasing researcher, with international part of the body cavity above the diaphragm housing the heart and lungs phosphorus The rise of sour sequencing technologies application and the development of commercialization pyrosequencing instrument, the pyrosequencing techniques application Fang Xing of China Do not end.But at this stage, there are several restraining factors in the application and popularization of domestic pyrosequencing techniques：(1) existing business Product pyrosequencing instrument such as PSQ96, GS20 is expensive；(2) commercialized pyrosequencing service latency length and It is very inconvenient；(3) although some laboratories are carrying out the research of the devices such as pyrosequencing chip, some laboratories at present There is pyrosequencing experimental rig homemade, simple in construction, but the country is not towards low side, cheap, commercialization The Sequence Detection Instrument based on pyrosequencing techniques, be restrict pyrosequencing techniques application development key issue.

Pyrosequencing system is carried out in micro environment, and usual reaction system is only in 50 μ L, required reaction bottom The amount of the reagents such as thing, DNA profiling and deoxynucleotide is very small；Meanwhile, the number of single sampling amount directly affects circulation The sustainability of reaction, excessive single sampling amount can make reaction solution volume become big rapidly, therefore cause template concentrations to decline It is too fast.Due to the non-linear increase of response delay that diffusion is produced, obtained fluorescent signals extend on a timeline, by force Degree is reduced on the vertical scale, and ultimately result in serious curtailment nucleic acid is sequenced length.It is generally acknowledged that caused due to follow-up sample-adding If reaction system increases within 10%, the influence to experimental result is within the acceptable range.If will be to one section The DNA segment of 20bp length is sequenced, then in 50 μ L reaction system, it is allowed to single sampling amount can only be no more than 0.3μL。

In addition, except sample-adding precision, sample-adding interlude accuracy is similarly important.Add only in constant duration Enter the dNTP needed for single cycle, can just make each residual dNTP palliating degradation degree suitable, then the shadow caused to lower secondary response Sound also will be equal.Benchmark could be provided to the signal in each cycle Deng the period, be easy to subsequently be calculated according to fluorescence signal intensity Nucleotides binding number purpose automated analysis.

Although the loading device of production domesticization is relatively abundanter, domestic existing micro sample-adding device has deficiency.Than Such as the sample-adding platform of Shanghai multiple day, as large automatic sample pool processing equipment, the sample-adding of the orifice plate of standard 96 can be carried out, shaken Swing, cleaning, but this set system is due to the limitation of nozzle processing technology, is loaded micro- precision minimum and there was only 1 μ L, it is impossible to meets The nL ranks of pyrosequencing requirement.By investigation, be limited to domestic application and manufacture level, the sample adding device of production domesticization all without Method meets the high request to sample-adding amount and repeatable accuracy in pyrosequencing.

Southeast China University Ge Jian emblems etc. disclose a kind of using faint light detection module and micro dNTP sample-adding modules as crucial mould The liquid phase pyrophosphoric acid analytical equipment of block, it is disclosed that an air pressure controls micro dNTP sample-addings module, can be achieved 96 road dNTP molten It is loaded while liquid, minimum sample-adding amount is 1.2 μ L, and worst error is 13%；But signal noise is larger, the improvement of a more step is still needed to (Ge Jianhui etc., the development of the gene assaying device based on pyrosequencing, Southeast China University, master thesis, 2006).

Wang Chunlin etc. discloses micro sample-adding system in the pyrophosphoric acid nucleic acid sequencing instrument of use piezoelectric ceramics shower nozzle a kind of, should Under driving stepper motor, 96 hole on-gauge plate samples can be carried out with the sample-adding in turn of 4 kinds of dNTP reagents, sample-adding repeats essence respectively Degree is more than 95%, and single sampling minimum can reach 0.l μ L.It is micro- used in above-mentioned two class preferably pyrophosphoric acid nucleic acid sequencing instrument Measure sample adding system structure more complicated, and sample needle is easily blocked up, dNTP by different modes spray into sequencing reaction liquid not with Reaction solution contact cause it is incomplete with the reaction of reaction solution undercompounding, it is also higher to dNTP required amounts and data are easily inaccurate； In addition, dismounting trouble, cost is high and is unfavorable for applying under specific condition.

Pyrosequencing (preosequencing) technology is a kind of new DNA sequence analysis skill developed in recent years Art, the pyrophosphoric acid that it discharges after being combined by nucleotides and template triggers enzyme cascade, promotes fluorescein luminescence and is examined Survey.It is a preferable genetic analysis technology platform, can both carries out DNA sequence analysis, the list based on sequence analysis can be carried out again Nucleotide polymorphisms (SNP) are detected and gene frequency is determined etc., and this technology has been widely used in medical biotechnology at present Etc. every field.

Pyrosequencing is by archaeal dna polymerase (DNA polymerase), adenosine triphosphate sulfurylase (ATP Sulfurylase), the enzyme of 4 kinds of same reaction systems of enzymatic of luciferase (luciferase) and bisphosphatase (apyrase) Chemiluminescence reaction is cascaded, reaction substrate is 5 '-phosphosulfate (adenosine 5 ' phosphosulfate, APS) and fluorescence Element.Reaction system also includes that DNA to be sequenced is single-stranded and sequencing primer.In each round sequencing reaction, a kind of dNTP is added, if should DNTP is matched with template, and polymerase can just be incorporated into primer strand and discharge the pyrophosphoric acid group of equimolar number (PPi).Sulfurylase is catalyzed ASP and PPi formation ATP, and the latter drives the fluorescein that luciferase is mediated to oxyluciferin Conversion, sends the visible light signal being directly proportional to ATP amounts, and by Pyrogram^TMBe converted into a peak value, its height with reaction The nucleotide number of incorporation is directly proportional.According to add dNTP types and fluorescence signal intensity can logging template DNA in real time core Nucleotide sequence.Atriphos (dATP) is replaced with effectively with the atriphos (dATP α S) of α-vulcanization in experimentation Utilized by archaeal dna polymerase, without being recognized by luciferin.Because SpdATP α S can reduce the concentration of dATP α S catabolites, In recent years, single-stranded DNA binding protein (single starnd DNA binding portein, SSBP) and purifying Spisomer This problem that dATPaS use dATP α S catabolites suppress bisphosphatase activity is preferably solved so that sequencing length Up to 10bp, application of the technology in genetic arts has been expanded.

In pyrosequencing, by using interim complementary strand synthesis reaction and chemiluminescence reaction, detection is luminous, so that To determine DNA sequence dna.By containing the reaction solution by the synthetically produced pyrophosphoric acid of complementary strand and remaining nucleic acid primer so as to carrying out The reactive tank of complementary strand synthesis is moved to other reactive tank, carries out luminescence-producing reaction, and pass through during reaction solution is moved The region for the enzyme for decomposing remaining nucleic acid primer is fixed with, after remaining nucleic acid primer is decomposed, pyrophosphoric acid is converted into ATP, Import chemiluminescence reaction groove.It is anti-to ensure but the drawbacks of prior art is to must be added to substantial amounts of substrate and enzyme is reacted It can should completely carry out, need to react away again afterwards and cleaned after unnecessary substrate, this not only adds course of reaction, increase every The cleaning of one step and reaction difficulty, can also cause very serious waste, the reaction time is long, waste of manpower thing to reagents such as substrates Power, virtually reduces pyrosequencing popularization in the market and uses potentiality.

The instrument applied to pyrosequencing is generally the monopolization manufacture of part producer and sold at present, and instrument is to match somebody with somebody with reagent Set sale, cost is sufficiently expensive during sequencing, and Measuring error process is both needed to depend on specific technical staff, and the cycle, long cost was high, Volume needed for reaction is larger, more increases the cost of reaction, testing result is unstable, and repeatable accuracy is low.Therefore design autonomous A DNA sequencer suitable for pyrosequencing is developed in research and development to be extremely necessary.

5th, the single-stranded isolation technics overviews of DNA

The single-stranded isolation technics of DNA is one of most common isolation technics in biomedical sector, it is adaptable to different nucleic acid samples The various scale sequencings of DNA of product and probe device, are widely used in biology, medicine and pharmacology, preventive medicine, zoology and botany, agriculture Animal husbandry, food and the field such as health, the energy and chemical industry, environmental monitoring and medical diagnosis and detection.In addition, suction single-stranded DNA It is attached, extract with isolation technics water quality, water source, biomaterial, biological fluid (such as blood, serum, blood plasma, cerebrospinal fluid, urine, Tear, sweat, digestive juice, seminal fluid, juice, tissue fluid, vomitus, excrement), tissue/cell and microbial lytic liquid, difference Analysis detection, separation and the purifying of the biologies such as albumen, the nucleic acid in source, chemical molecular and medicine etc. and oligonucleotides, many It is widely used in terms of the synthesis of peptide, lead compound and medicine, it is closely bound up with daily life, in biological medicine Field has very important status.

The single-stranded separation methods of DNA and the deficiency of presence commonly used in biomedicine field are as follows：

1. thermal denaturation or alkali process.This kind of method is mainly heated double stranded PCR products or alkali process, due to DNA Double-strand hydrogen bond under high temperature or a certain degree of alkaline environment can be broken so that DNA is changed into single-stranded.Although this kind of Method And Principle can OK, it is simple to operate, but this kind of method is due to its separation rate and purity is relatively low and the gradually purifying not as single stranded DNA, is used for DNA double-strand separation.

2.T7 reverse transcription methods.This kind of method is to add T7 promoters at an end of PCR primer 5 ', to purify PCR amplification productions Thing is template, with the synthesizing single-stranded RNA of the external reverse transcription of t7 rna polymerase (Hughes, et.al., Nat.Biotechnol., 2001,19:342-347).Although this kind of method principle is feasible, the single-stranded separation rates of DNA are higher, and obtain the single-stranded purity of DNA compared with Height, but whole separation process need to be divided into two big steps completions, and operation is inconvenient, and the time is longer, and need to strictly control the dirt of RNase Dye, therefore with certain limitation.

(Higuchi and Ochman, Nucleic.Acids Res., 1989,17 3. Exonucleolytic enzyme process:5865).By It is phosphorylated in a PCR primer, PCR primer is when using exonuclease digestion, and the primer amplification chain being phosphorylated is not cut Cut, enzyme is heated inactivation after digestion.This kind of method also needs purified pcr product, and separable programming is tediously long, and operation is also inconvenient, and And the single-stranded pick-up rates of DNA depend on the activity of excision enzyme, uncontrollable factor is too strong, and the stability of experimental result is inadequate；Therefore, should The implementation rate of the method for kind is not wide, and versatility is not high.

4. denaturing high-performance liquid chromatography (denaturing high-performance liquid Chromatography, DHPLC).Under conditions of partial denaturation, pass through heterozygosis and zygoid retention time in post Difference, finds DNA mutation.Allogeneic dna sequence DNA double-strand is different from the melting properties of homologous dna double-strand, heterologous under the conditions of partial denaturation Double-strand is because having the presence of mispairing district and more mutability, and retention time in the chromatography column is shorter than homoduplex, therefore is first eluted down Come, bimodal or multimodal elution curve is shown as in chromatogram.Because a PCR primer is by biotin labeling, its PCR amplifications Chain in DHPLC, can and another common chain separate (Dickman and Hornby, Anal.Biochem., 2000,284: 164-167).DNA needed for this method can directly be obtained in 15min from double stranded PCR products is single-stranded, but this kind of method Implement to need supporting sufficiently expensive instrument, therefore be difficult to popularize all the time.

5. magnetic capture method.The surface of superparamagnetic nano particle is improved with nanometer technology and surface modification Afterwards, it is prepared into superparamagnetism silica nanometer magnetic bead.The magnetic bead can specifically be recognized with nucleic acid molecules on micro interface and Efficiently combine.Using the superparamagnetism of silica nanoparticle, in Chaotropic salt (guanidine hydrochloride, guanidinium isothiocyanate etc.) and outside Plus in the presence of magnetic field, can be separated from the DNA and RNA in blood, animal tissue, food, pathogenic microorganism equal samples, so Target to be obtained with NaOH processing single-stranded afterwards.This kind of method is simple to operate, the used time is short, and whole flow of extracting is divided into four steps, mostly may be used To be completed in 36-40 minutes, and safety non-toxic, without using toxic reagents such as the benzene in conventional method, chloroforms, to experimental implementation The injury of personnel is reduced, and meets modern environmental protection concept, and the magnetic bead specific binding single-stranded with DNA make it that the DNA extracted is single-stranded pure Degree is high, concentration is big, but the coating magnetic bead used in this kind of method is costly, and needs, by magnetic frame separation, not only to separate Cost is higher, also inconvenience, therefore limits the popularization of the technology to a certain extent.

6. asymmetric PCR.Above method is both needed to after PCR carry out extra processing, and asymmetric PCR can be expanded in PCR While prepare DNA it is single-stranded.Conventional asymmetric PCR is normally expanded using the primer of two inequalities in the circulation of beginning Increase.With the increase of circulation, measure few primer and gradually exhausted, and can to continue straight line amplification generation DNA single-stranded for the primer of excess (Gyllensten and Erlich,Proc.Natl.Acad.Sci.U.S.A.,1988,85:7652-7656).This kind of method With higher hybridization sensitivity and specificity, and ease-to-operate is stronger, but the ratio of its primer needs optimization, and non-spy The chance increase of different amplification, in addition, the single-stranded separation processes of DNA need to rely on electrophoresis, separable programming is complicated, and electrophoresis is often visible Smear, it is time-consuming and is not obvious.

Above-mentioned several separation methods are respectively provided with certain limitation, therefore, in order to meet to the operable of the single-stranded separation of DNA Property and cost-effectiveness requirement, DNA of the prior art is single-stranded to use integrated extraction work station, by with streptavidin Affine connector is combined with DNA double chain, and work station has suction filtration pin and supporting pump, and the affine connectors of DNA with reference to after pass through Suction filtration absorption filter membrane bottom in suction filtration pin, work station is furnished with track and related system, and suction filtration pin is moved to Sheng by suction filtration after terminating Have in NaOH disk, by alkali process solution double helix, it is single-stranded to obtain DNA, clean collect after suction filtration again.General 24 of suction filtration pin (4*6) is one group, and the sample or reagent of sufficient amount are must assure that when using to ensure the normal operation of work station, therefore this Collection mode single-stranded DNA is very dumb, can only add work station with fixed amount and be operated, and substantial amounts of loss is more Produced in secondary suction filtration and transfer process, this is very unfavorable to micro-collection, and suction filtration pin group needs to be operated simultaneously, this All there is certain volume requirement to each part of work station, whole work station space-consuming is very big.Huge system causes in DNA In single-stranded separation operation process, micro- splitter needs liquid relief back and forth, and operation is very cumbersome, not only separation cycle length, efficiency It is low, and integral device is expensive, causes the costly of the single-stranded separation of DNA, in addition it is also necessary to substantial amounts of reagent and other resources are expended, It is extremely uneconomical.In addition, suction filtration pin is metal material in the work station, and it is expensive, re-used after often handling, therefore easily Cause the cross pollution between residue, reliability is not high, the accuracy to separation and testing result can cause certain do Disturb and influence.And it is adherent that portion of residual solution is had during solution extraction so that a certain amount of target DNA is single-stranded can not be micro- Splitter is adsorbed, and is caused the DNA proportion of single chain reduction obtained, be have impact on separation rate, cause waste.Therefore, for pyrophosphoric acid The single-stranded separation problems of DNA of the high quality and high efficiency of sequencing are urgently to be resolved hurrily.

The content of the invention

In view of the above-mentioned problems existing in the prior art, it is an object of the invention to provide a kind of easy to operate, quick detection DNA sequencing apparatus and system based on pyrosequencing.

For achieving the above object, the technical solution adopted by the present invention is as follows：

A kind of DNA sequencing device based on pyrosequencing, including sample area, reaction zone and detection zone；

The sample area includes rotatable separator disk, and the separator disk is located at the top of reaction zone, the separator disk bag At least one DNA Disengagement zone is included, the Disengagement zone includes the internal hollow Filter column for being provided with filter membrane, is connected with affine connector DNA is single-stranded and is not connected with the single-stranded separation after the membrane filtration of the DNA of affine connector；

The reaction zone includes sample-adding portion and sample cell, and the sample-adding portion includes dNTP reagent troughs, sample-adding punch block and installation Multiple sample needles thereon, the separator disk is located above the side of reagent trough；The sample-adding punch block is located on sample cell；It is described Sample cell is provided with multiple grooves position；

The detection zone includes bioluminescent detection device, and the groove position is connected with bioluminescent detection device；

The sample area, reaction zone and detection zone are arranged in analysis station, and the side of the analysis station, which is provided with, to be used to operate Manipulator.

It is used as the further improvement of above-mentioned technical proposal：

Preferably, filter membrane is located at one end of Filter column.In other words, filter membrane constitutes the bottom of Filter column, DNA Disengagement zone Filtering surface is located at splitter bottom, that is to say, that the single stranded DNA after separation is on filter membrane.

Further, as a preferred embodiment, the external diameter of Filter column is equal to the internal diameter of collecting pipe, frictional force is passed through Filter column can be made to be maintained at a relatively steady state with collecting pipe, it is not necessary to which plus structural is that Filter column progress is spacing solid It is fixed.

Another preferred mode, the separator disk is provided with multiple collecting pipes, and the Filter column is installed in collecting pipe, institute State the non-end that filter membrane is located at Filter column.

Further, the filter membrane is high molecular nano-microsphere structure, and the aperture between the high molecular nano-microsphere is less than parent With the diameter of connector.

Further, the collecting pipe is provided with dismountable upper lid, and the upper lid is connected by connect band with collecting pipe.

Single stranded DNA after separation is to be connected with the single-stranded of affinity body, it is necessary to which this chain can be washed to filter membrane on filter membrane It is de-, another complementary strand without affine connector is contained in filtrate, it is necessary to can enter to the single stranded DNA in filtrate during backward sequencing Row is collected.When needing to collect a chain of the band affine connector on filter membrane, collecting pipe can use the collecting pipe reclaimed, Now collecting pipe only plays a part of Recycling of waste liquid, and reclaiming to use reduces cost, and environmental protection reduces the generation of white pollution.

Preferably, the reagent trough is provided with multiple reaction positions, and the side-lower of the reagent trough is provided with substrate supply unit, described Substrate supply unit conveys dNTP tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP by conveyance conduit to reaction position.

Preferably, the sample-adding punch block is disc, and the sample-adding punch block can be along reagent trough and sample cell by move portion Between move reciprocatingly；The sample-adding punch block is rotated by rotating shaft.

Preferably, the move portion includes being fixed on the slide rail and the sample-adding punch block above analysis station by mounting bracket The sliding block of bottom.

Preferably, the rotating shaft is provided with motor.

Preferably, the sample needle is solid needle, and the sample needle is fixed in the through hole of sample-adding punch block.

Preferably, the sample-adding portion also includes dry slot and rinse bath.

Preferably, the groove position is evenly distributed in a series of concentric circles along the center of circle of sample cell, and the groove position is by transparent material Texture is into the groove position extends into bioluminescent detection device.

Preferably, the bioluminescent detection device includes set casing and camera, and the camera is rotatable to be located at fixation In shell, the camera is located at all grooves position and enclosed on the axis set.Camera can shoot the reaction in all groove positions by rotation Situation.

The present invention also aims to there is provided a kind of DNA sequencing system based on pyrosequencing, including above-mentioned survey Sequence device, and for controlling the control zone of sequencing device；The control zone includes microcomputer, Optimizing Control System, position Control system, environmental control system, activity monitor system and driving control system；The environmental control system includes dense to solution The control of degree and pH value.

In view of the above-mentioned problems existing in the prior art, the of the invention DNA sequencing apparatus and system based on pyrosequencing Advantage is：

(1) extracted DNA is single-stranded by way of filter centrifugation, the economization for realizing a sample one collection is small There is provided a kind of single-stranded separation method of rapid DNA and device of efficiently low loss for typeization collection.This method is simple to operation, obtains Target sample time short efficiency high, the collection single-stranded available for trace amount DNA and extraction are taken, sample loss can almost be ignored, make With reagent is few, requirement to equipment is low, in separation process without suction pump, device configuration is enormously simplify, equipment is reduced total Cost, is effectively simplified operating procedure, shortens the operating time, reduces working strength, improves operating efficiency；Using with it is normal The supporting Filter column of rule centrifuge tube not only ensure that the quality and efficiency of separation, also separation process simply easily be realized, filter The filter membrane of post is separated by the way that physics mode is single-stranded to DNA, reduces difficulty and the condition requirement of separation.Filter membrane is by homogeneous Material with identical structure composition, can two-way exchange use, increase the designability of Filter column, can not only be made as one end Hollow structure with filter membrane, enriches the structure and species of the single-stranded separators of DNA, on the premise of identical function is ensured, increases The application occasion of the DNA single-stranded separators of the present invention is added, it is to avoid product structure is single, and adaptability is significantly increased；And Structure symmetrical above and below can be used, can up and down exchange and use cooperatively.The single-stranded separators of this DNA are moulded using cheap PC Material, economic and practical, the design of infundibulate inner chamber is conducive to the aggregation and guiding of reaction solution so that reaction solution separation is more abundant More completely, the target DNA for obtaining higher proportion is single-stranded, it is ensured that higher separation rate, it is to avoid waste.In addition, this DNA is single Chain separation device applies also for commercially available centrifuge tube, standardized designs so that the single-stranded separator versatilities of this DNA are extremely strong, is applicable Face is extremely wide, therefore its application prospect is very wide.

(2) present invention is the micro to sample of achievable dNTP reagents using a sample needle simple in construction, has abandoned transmission Hollow needle tube take out spray formula give sample loading mode, only by the absorption affinity between the sample needle and liquid of the sample needle and its in difference The control of translational speed difference in liquid be can be achieved microsampling sample-adding, and only by sample needle in sequencing reaction liquid on Lower motion is that stirring can be achieved to cause the more complete result of enzyme reaction accurate for several times, and this loading methods and its device are suitable for any In detection means, dismounting is easy, cleans simple and convenient, and will not occur a variety of sample adding devices of the prior art such as stifled pin not Foot, sample-adding repeatable accuracy is more than 95%, and minimum sample-adding amount, up to 0.1 μ L, sample-adding homogeneity is good, the sequencing time shorten dramatically and As a result accuracy is high；In addition qualitative and quantitative detection can be carried out to sample nucleic acid sequence by detection means associated with institute；Therefore Its application prospect is very wide.

(3) the DNA sequencing device based on pyrosequencing of the invention, whole process is manipulated using manipulator, no infection, Accuracy rate is high, improves know clearly efficiency and precision.

In a word, the DNA sequencing apparatus and system based on pyrosequencing that provides of the present invention reduces substrate and enzyme system Consumption, detection is accurate, and reaction speed is fast, integrated height, and can detect one or more SNP site and single stranded DNA piece simultaneously Section, enzyme system and substrate have broken the monopolization of part producing business, and price is greatly reduced, analytical equipment precise structure, to DNA to be measured The consumption of fragment and substrate requires low, reduces the testing cost of pyrosequencing, expands the use scope of pyrosequencing.

Brief description of the drawings

Fig. 1 is the structural representation of the DNA sequencing device based on pyrosequencing of the present invention.

Fig. 2 is the control schematic diagram of control zone of the present invention.

Fig. 3 is one to use preferred embodiment provided by the present invention for the DNA Disengagement zone of pyrophosphoric acid.

Fig. 4 is the structural representation of Filter column in the embodiment of the present invention 1.

Fig. 5 is the structural representation of Filter column in the embodiment of the present invention 2.

Fig. 6 is the structural representation of Filter column in the embodiment of the present invention 3.

Fig. 7 is the structural representation of Filter column in the embodiment of the present invention 4.

Fig. 8 is provided by the present invention for the sample needle schematic diagram in the sample adding device structure of pyrosequencing system.

Fig. 9 is that the sequence spectrum that measures of sample adding device provided by the present invention for pyrosequencing system is used in embodiment Figure.

Label declaration in figure：

1st, conveyance conduit；2nd, separator disk；21st, Filter column；211st, split tunnel；22nd, filter membrane；23rd, collecting pipe；231st, on Lid；232nd, connect band；3rd, sample cell；31st, groove position；4th, it is loaded punch block；41st, sample needle；42nd, rotating shaft；5th, bioluminescent detection is filled Put；51st, set casing；6th, analysis station；61st, mounting bracket；62nd, slide rail；7th, reagent trough；71st, dry slot；72nd, rinse bath；73rd, substrate Supply unit；74th, position is reacted.

Embodiment

The present invention is made with reference to embodiment further in detail, intactly to illustrate.

Embodiment 1

Fig. 1 to Fig. 3 shows the first embodiment of the DNA sequencing system of the invention based on pyrosequencing.This hair The bright DNA sequencing device based on pyrosequencing, DNA sequence dna, DNA sequence dna to be sequenced are tested and analyzed available for pyrosequencing For target sequence, pyrosequencing is carried out after target DNA sequence is expanded.

The DNA sequencing system based on pyrosequencing of the present embodiment includes DNA sequencing device and control zone, DNA sequencing Device includes sample area, reaction zone and detection zone.Sample area, reaction zone and detection zone are arranged in analysis station 6, sample area, anti- Area and detection zone is answered to monitor, control by control zone.Whole analysis process uses Robot actions.

, it is necessary to first be expanded to the DNA profiling with sequencing before carrying out pyrosequencing, to reach amplification requirement DNA concentration.When amplimer is designed, affine connector is carried on amplimer, affine connector is preferably biotin, affine company Junctor is preferably marked on one end primer of target DNA, and PCR amplifications are carried out using prior art.

Sample area includes separator disk 2.Separator disk 2 is located at the top of reaction zone, and after target DNA amplification, target DNA is double-strand DNA, pyrosequencing needs single stranded DNA, and separator disk 2 includes at least one DNA Disengagement zone, and DNA Disengagement zone uses physical filtering Mode, Disengagement zone includes the internal hollow Filter column 211 for being provided with filter membrane 22, and filter membrane 22 is high molecular nano-microsphere structure, It is because the aperture between high molecular nano-microsphere is less than the diameter of affine connector, the DNA marked with affine connector is single-stranded Stay on film, need the DNA for collecting tape label single-stranded or its complementary strand according to sequencing, for pyrosequencing.

In the present embodiment, separator disk 2 is provided with multiple collecting pipes 23, and Filter column 21 is installed in collecting pipe 23, filter membrane 22 End in one end of Filter column 21.The external diameter of the hypomere of Filter column 21 is identical with the internal diameter of collecting pipe 23.

Double-stranded DNA stays in a chain for treating affine connector on filter membrane 22 after alkaline hydrolysis, and collecting this chain only need to be in mistake Filter to add in post 21 after collection liquid and collect；Such as need to collect complementary strand, the complementary strand in filtrate is received after can filtrate be collected Collection.

As shown in figure 3, need to topple in time after the waste liquid that produces, each step when collecting pipe 23 is used to collect centrifugation to prevent Only cross pollution, the upper pipes of collecting pipe 23 are cone as cylinder, lower end, and bottom is dome shape.As another excellent The embodiment of choosing, the upper lid 231 of the configuration of collecting pipe 23, upper lid 231 is detachably connected to collecting pipe 23 by connect band 232 On, due to the connection of connect band 232, loading upper lid 231 after Filter column 21 also can closely be fastened on Filter column 21 or collecting pipe 23 On, the effect of upper lid 231 is to prevent liquid splash from going out Filter column 21 and causing damage and pollute when liquid is centrifuged.

The preferred material of filter membrane 22 is that hole is preferably 10 μm between polyethylene microballoon, its microballoon in the present embodiment, is less than The diameter of affine connector, will directly can be stayed on film by physical filtering with the single-stranded of affine connector, be filtered off not connected One chain of affine connector, its absorb-elute effect is good, and the DNA rate of recovery is high, low in raw material price environmental protection.

As shown in figure 4, in order to which the filter membrane 22 in the present embodiment is not moved in piping and druming or centrifugal process, the present embodiment In further preferably be provided with film pressing device in the top of filter membrane 22, film pressing device includes pad and/or press mold frame.The liquid of purifying to be separated Body is first passed through after pad and contacted with filter membrane 22, and pad is preferably fibrous material, soda acid and most of organic solvent is can tolerate, to big Partial biomolecule will not produce absorption.

Preferably in the top of pad, the side not contacted with filter membrane 22 is additionally provided with press mold frame, press mold frame and Filter column 21 Body material is identical, and filter membrane 22 is compressed by mechanical pressure, filter membrane 22 is not moved during the use such as piping and druming or centrifugation It is dynamic, cause to collect and lose.

Reaction zone includes sample-adding portion and sample cell 3, and sample-adding portion includes dNTP reagent troughs 7, dry slot 71, rinse bath successively 72nd, sample-adding punch block 4 and the multiple sample needles 411 being installed on sample-adding punch block 4.Separator disk 2 is located above the side of reagent trough 7.Plus Sample punch block 4 is disc, and sample-adding punch block 4 can be moved reciprocatingly by move portion along between reagent trough 7 and sample cell 3.Move portion Including being fixed on the slide rail 62 of the top of analysis station 6 and the sliding block of sample-adding punch block 4 bottom by mounting bracket 61.Dry slot 71 is true Empty dry section.

Reagent trough 7 is provided with substrate supply unit 73, substrate supply unit 73 provided with four reaction positions 74, the side-lower of reagent trough 7 DNTP different nucleic acid primers are conveyed to reaction position 74 by conveyance conduit 1.Reaction position 74 to be measured is used to hold DNA to be sequenced Sequence, substrate supply unit provides dNTP, and with target DNA hybridization reaction, environmental basis is provided for hybridization reaction.For pyrosequencing DNTP include tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP, substrate be used for target DNA hybridize, and need reaction Related enzyme systems catalytic reaction is added in system to carry out, specially archaeal dna polymerase, carry out complementary strand synthesis reaction, closed in complementary strand Into when obtained accessory substance pyrophosphoric acid change into ATP, react ATP and luciferin in the presence of luciferase, lighted.Such as Complementary strand synthesis occurs for fruit, then produces pyrophosphoric acid, as a result light, therefore by being monitored to it, can learn that generation is complementary Chain is synthesized, i.e., the base species that group enters thereby determines that DNA sequence dna.

Sample-adding punch block 4 is rotated by rotating shaft 43, and rotating shaft 43 is provided with motor.Sample needle 411 is solid needle, is used In transfer dNTP reagents, sample needle 411 is fixed in the through hole of sample-adding punch block 4.By DNA it is single-stranded according to reaction system the need for measure It is transferred in reaction position 74, sequentially adds dNTP after adding enzyme system, addition sequence is not limited, and for each site, four kinds of substrates are each Add once, substrate is provided by substrate supply unit 73.

Detection zone includes bioluminescent detection device 5, and groove position 31 is connected with bioluminescent detection device 5.As closed after reaction Into complementary strand, then light, bioluminescent detection device 5, which is used to detect, judges whether the site is reacted and lighted, and then sentences Break and the base sequence in the site.

Punch block 4 is loaded to be located on sample cell 3；Sample cell 3, which is provided with multiple grooves position 31, groove position 31, sequencing reaction liquid. Groove position 31 is evenly distributed in a series of concentric circles along the center of circle of sample cell 3, and groove position 31 is made up of transparent material, and groove position 31 is extended into Bioluminescent detection device 5.Bioluminescent detection device 5 includes set casing 51 and camera, and camera is rotatable to be located at set casing In 51, camera is located on the axis that all grooves position 31 is gathered.Camera is commercially available iKon-M deep refrigeratings image CCD camera, and Connection computer and the spectrogram that detection is presented.

As shown in figure 8, the end face of sample needle 41 is semicircular body；The sample of known array is detected using above-mentioned sample adding device (sequence is CAATATTCGCCAGGT), the wherein a diameter of 1.5mm of sample needle 41, surface smoothness are Ra 0.8；Loading methods bag Include：Step a, the dNTP reagents for inserting sample cell 3 by sample needle 41 cause the periphery of sample needle 41 to adhere to the dNTP reagents；Step Rapid b, will adhere in the insertion groove of sample needle 41 position 31 of the dNTP reagents, then sample needle 41 is left sequencing reaction liquid；It is described Translational speed when sample needle 41 described in step a leaves the dNTP reagent solutions is 10cm/s, is loaded described in the step b Translational speed when pin 41 leaves the sequencing reaction liquid is 1cm/s, and sequencing is left after being moved up and down 8 times in sequencing reaction liquid Reaction solution.

Fig. 9 is the sequencing spectrogram obtained by the use pyrophosphoric acid nucleic acid sequencing detection means of the present embodiment, as shown in Figure 9, wherein DNTP order TCATATTCGCCAGT is sequentially added, wherein non-appearance when adding T first, and be not inconsistent with actual sequence subsequently DNTP reagent solutions also non-appearance (being marked in Fig. 9 with circle, i.e. T, T, C), its sequence measured is CAATATTCGCCAGGT, Completely the same with sample actual sequence, accuracy is 100%, detects that the time of single base is less than 1.5 minutes, this sequence is about It is time-consuming 25 minutes, and minimum sample-adding amount is up to 0.1 μ L.

The set-up mode of the DNA sequencing system based on pyrosequencing of the present invention can preferably transmit substrate and DNA, Reduce the loss in transmittance process.

The shape of reaction zone is not limited, the concrete structure in analytical equipment can according to being designed adjustment the need for detection, Any planform and relative position that may realize pyrosequencing is considered as falling under the scope of the present invention.

Substrate is loaded into reaction zone, now added that DNA to be measured is single-stranded in reaction zone and other required reaction systems in Reagent, is calculated with the amount of 5ul needed for detecting, and reaction system is as follows：

Enzymatic mixture includes：

Substrate mixture includes：

APS 0.4mg/L；

Firefly luciferin 0.4mmol/L.

In course of reaction, react, needed after reaction pair in each step selection optimum pH value for enzymatic activity PH value in reaction system is adjusted to adapt to the reaction condition ordinary skill in other reactions, specific reaction system Personnel can draw according to prior art.Include apyrase in washing step to remove excessive nucleosides for example, working as Sour species and during ATP, because the continuity of process step, buffer solution can be used together with apyrase, its pH Can optimize the clean property level of apyrase.Then, polymerase is used in next nucleotides incorporation step, The different buffer solutions with the optimal pH condition for polymerase can be used, to optimize the clean property of polymerase.In addition, every Planting optimized buffer liquid may include the preferred counter ion counterionsl gegenions for every kind of enzyme, for example, buffered for apyrase Ca for liquid²⁺With the Mg for polymerase buffer²⁺。

As shown in figure 9, in reaction system, detection sequence is CAATATTCGCCAGGT, wherein sequentially adding dNTP order For TCATATTCGCCAGT, wherein non-appearance when adding T first, and the dNTP reagents not being inconsistent with actual sequence subsequently also do not go out Peak, its sequence measured is CAATATTCGCCAGGT, and completely the same with sample actual sequence, accuracy is 100%；Detection is single The time of individual base is less than 1.5 minutes, and this sequence about takes 25 minutes, and minimum sample-adding amount is up to 0.1 μ L.

Sequencing result is judged that other can detect that the device of bioluminescence can be used for inspection by bioluminescence Device is surveyed, is not limited herein.

The DNA sequencing device based on pyrosequencing based on pyrosequencing in embodiment of the present invention, is also wrapped Liquid supply and the passing away between each sample storage device are included, liquid supply passage is used for the reagent and sample for conveying question response Product, liquid discharge passage is expelled to collecting part for that will react the liquid terminated afterwards or after centrifugation.

Control zone includes microcomputer, Optimizing Control System, position control system, environmental control system, Activity determination system System and driving control system.Environmental control system includes the control to solution concentration and pH value.

Carried out in the reaction, it is necessary to ensure that each portion is operated in the environment of optimum in the analytical equipment of the present invention, enzyme System needs to be reacted in active highest reaction system, to ensure that reaction is complete, therefore be provided with control zone to ensure Some components of effective progress, including centrifuge, pH meter, heating component, control signal transmitting assembly are capable of in these reactions Deng the standing structure of sequencing equipment.

The system and method for embodiment of the present invention may include to carry out some designs using computer-readable medium, divide Analysis or other operations, such media storage have the instruction for performing on the computer systems.For example, processing detects signal And/or analysis some embodiments of the data of sequencing result system and method generation, wherein processing and analysis embodiment Perform on the computer systems.

An exemplary for the control zone of the present invention may include any kind of computer platform, for example Work station, personal computer, server or any other existing or future computer.Computer generally includes oneself and knows part, Such as processor, operating system, system storage, memory storage (memory storage device), input and output control Device processed, one output device of input and display.Person of ordinary skill in the relevant will be understood that have many possible computers to match somebody with somebody Put and part, and may also include each part unit of high-speed memory, data and many other devices.

Display may include the display for providing visual information, and such information is generally logically and/or physically organized As pel array.Interfacial level controller is may also comprise, wherein may include that any different oneself knows or following software program, is used for Input and output interface are provided.For example, interface may include so-called " graphic user interface (Graphical User Interfaces, commonly referred to as GU I) ", it can provide the user one or more images and show.Use the common skill of association area Oneself selection for knowing of art personnel or input mode, interface usually can receive user's input.

In identical or alternate embodiment, application on computers can be used and include so-called " Command Line Interface " and (lead to Frequently referred to CLI) including interface.The commonly provided texts based on application with user's interphase interaction of CLI.Generally, order line circle Face is by the way that display is with line of text form display output and receives input.For example, some implementation procedures may include so-called " order Oneself Unix command line interpreters (Unix for knowing of row interpretive program (shell) " such as person of ordinary skill in the relevant Shell), or using object oriented program architecture Microsoft Windows Powershell, for example Microsoft.NET frameworks.

Person of ordinary skill in the relevant will be appreciated that interface may include one or more GUI, CLI or its combination.

Processor generally performs operating system, and any operating system of the prior art may be selected in operating system, as long as this Art personnel can be used for the work such as processing detection result and data to think to can be included within protection domain.

The DNA sequencing system based on pyrosequencing based on pyrosequencing in the present invention can be widely used for Determination, diagnosis, the inspection of DNA sequence dna, determination, diagnosis of SNP site etc., it can be achieved to be sequenced while certain sample, principle pair The condition of sequencing requires low, it is not necessary to the expensive experiment reagent of extra increase excitation source and fluorescer etc., can be achieved easy to be honest and clean The DNA sequencing work of valency, and testing result is stable, and accuracy rate is high, and overcoming conventional equipment can not fully be carried out or complementary The drawbacks of chain synthesis reaction is first excessively carried out, reaction volume is small, and the work of the stage of reaction can be completed within the shorter time.

The analysis method of the DNA sequencing apparatus and system based on pyrosequencing provided using the present invention, including it is following Step, wherein undisclosed partly can refer to prior art：

(1) combine：DNA fragmentation after amplification is combined with sepharose 4B, the length of DNA fragmentation is 10~20kb, DNA minimums applied sample amount should be not less than 500ng, a diameter of 30 μm of sepharose 4B, and surface is covered with biotin and Streptavidin, amplification DNA fragmentation afterwards is spontaneously specifically bound with the coated sepharose 4B of Streptavidin；

(2) centrifuge：The DNA double chain after combining is drawn into Filter column 21, Filter column 21 is previously charged into collecting pipe 23, is put Enter after centrifuge with 12000rmp centrifugation 1min, remove excess of solvent；Wherein collecting pipe 23 is 1.5mL centrifuge tubes；Filter column 21 For upper end open, the semi-enclosed cylinder in lower end, the upper end open outer rim of Filter column 21 is provided with boss, to be fixed on collecting pipe 23 On, the lower end of Filter column 21 is built-in with filter membrane 22, a diameter of 4.5mm of Filter column 21, a diameter of 4.7mm of filter membrane 22, by that will filter Film 22 is pressed into be allowed to be brought into close contact in Filter column 21 by force and left no gaps；

(3) wash：Appropriate 70~80% ethanol is being added, the amplifing reagents such as the Taq enzyme of residual are washed away, gently blown and beaten mixed Even rear 12000rmp centrifugations 1min；

(4) alkaline hydrolysis：Add 0.4M NaOH and 1M NaCl and untie double-stranded helical, 12000rmp is centrifuged after gently piping and druming is mixed 1min；

(5) pH value is adjusted：Appropriate elution buffer or ultra-pure water cleaning are added, the NaOH of residual is washed away, equilibrium ph is into Property, 12000rmp centrifugations 1min after gently piping and druming is mixed；

(6) collect：Collection liquid such as ultra-pure water etc. is added, gently blows and beats to the single-stranded complete suspensions of DNA, is used after sucking-off after addition In pyrosequencing or 4 DEG C of sealing preserves.

(7) it is loaded：Any dNTP reagents are dipped by sample needle 41 so that the attachment of the periphery of the sample needle 41 is described DNTP reagents；

(8) react：The sample needle 41 for adhering to the dNTP reagents is inserted in sequencing reaction liquid, then makes the sample needle 41 Leave the sequencing reaction liquid；

(9) conclusion：Bioluminescent detection device 5 connects a computer, takes pictures and present the spectrogram of detection.

Wherein, be realize complete sequencing, be preferably by 4 kinds of dNTP reagents it is any one or more depending on sequence to be measured with which kind of Order is added in sequencing reaction liquid, for example：Only detect whether single base becomes the different time, can first be determined according to known context After base positions to be measured, 4 sample-addings of above-mentioned steps (adding 4 kinds of dNTP reagents) are repeated in, same sequencing reaction is separately added into The base is detected in liquid.

Because the diameter of affine connector is more than the diameter of filter membrane 22 of the single-stranded separators of DNA, therefore pass through DNA is single-stranded During filter membrane 22, the DNA of uncombined affine connector is single-stranded and other impurities can be by filter membrane 22, and combines affine connector Single-stranded be left on film and can not pass through, this filtering is physical.

Involved solution, parameter and other separation conditions, are the present embodiment in DNA separation processes in the present embodiment In preferred embodiment, it is any to play experiment condition, parameter and the solution of respective action with reference to prior art and can use The involved design parameter isolated and purified in process, the present embodiment and solution selection should not be used as to the present invention in the present invention Limitation.

Sequencing spectrogram obtained by the present embodiment and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) It is the uniformity in the range of theoretical error with the data obtained of embodiment 1, it is 96% that it, which is loaded repeatable accuracy,.

Embodiment 2

Because the chain collected on filter membrane 22 needs to be suctioned out or pours out, such collection mode is likely to result in one Fixed collection loss, therefore Filter column 21 is preferably set to the bipitch structure that two exchange is used by inventor, filter membrane 22 is set In Filter column 21 non-end face, it is necessary to when collecting, usual manner is used after the two of Filter column 21 is exchanged, such as centrifugation, It can elute all DNA on filter membrane 22 is single-stranded, reduce sample loss.

As shown in figure 5, the present embodiment and embodiment 1 are differed only in, the Filter column 21 in embodiment 2 is symmetrical above and below The hollow form cylinder of structure, filter membrane 22 is vertically positioned in the middle part of hollow cylinder, the Filter column 21 can two ends exchange and use, hollow posts The internal diameter of body is 4.5mm, a diameter of 4.7mm of filter membrane 22, is allowed to be brought into close contact by the way that filter membrane 22 is pressed into Filter column 21 by force Leave no gaps, the spacing squeeze-film mechanism (not shown) of filter membrane 22 is added in cylinder, displacement can not be carried out to fix filter membrane 22.By In the single-stranded separators of the DNA either still functionally two ends all same in structure, thus the single-stranded separators of the DNA without Liquid feeding end and outlet end need to be distinguished, two ends can arbitrarily select one and use when in use.

After unnecessary NaOH is washed away by step (4), the two ends of Filter column 21 are exchanged, collection liquid is added, it is static More than 1mins, elution step can be carried out without piping and druming, and centrifuge speed is no more than 10000rpm during elution, at least centrifuges 2min.DNA concentration can be detected by nanodrop after collection, carry out pyrosequencing.

Sequencing spectrogram obtained by the present embodiment and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) It is the uniformity in the range of theoretical error with the data obtained of embodiment 1, it is 95% that it, which is loaded repeatable accuracy,.

Embodiment 3

As shown in fig. 6, the present embodiment and embodiment 1 are differed only in, the mistake of the single-stranded separators of DNA in embodiment 3 Filter post 21 and use space circular platform type structure, filter membrane 22 is arranged at the public top surface of two circular platform type Filter columns 21, circular platform type filtering The top surface diameter of post 21 is consistent with the diameter of filter membrane 22, and the both sides of filter membrane 22 are provided with squeeze-film mechanism (not shown), with fixed filter Film 22 can not carry out displacement.The side wall of the open lower end of truncated cone-shaped Filter column is provided with boss, it is ensured that timing can be consolidated at two ends On the boss for being scheduled on collecting pipe 23.

The step of using the separation method of separator affiliated in the present embodiment with described in embodiment 1, is identical, with reality Apply differing only in for example 1：After unnecessary NaOH is washed away by step (4), the two ends of Filter column 21 are exchanged, adds and collects Liquid, static more than 1mins can carry out elution step without piping and druming, and centrifuge speed is no more than 10000rpm during elution, at least Centrifuge 2min.DNA concentration can be detected by nanodrop after collection, carry out pyrosequencing.

Embodiment 4

As shown in fig. 7, the present embodiment and embodiment 1 are differed only in, in order to preferably assemble with guiding reaction solution, make Reaction solution separation more fully more completely, evades filtering dead angle, and the embodiment is additionally arranged a split tunnel on the basis of embodiment 1 211, the split tunnel 211 is arranged between two Filter columns 21 and connected with the two, two Filter columns 21 and split tunnel 211 Concentrically axis, filter membrane 22 is arranged on the plane of symmetry of split tunnel 211, and the both sides of filter membrane 22 (do not show provided with squeeze-film mechanism in figure Go out), displacement can not be carried out to fix filter membrane 22, the middle part of two Filter columns 21 is provided with boss, it is ensured that two ends can be consolidated to timing On the boss for being scheduled on collecting pipe 23.

One end that Filter column 21 is connected with split tunnel 211 is rounding mesa-shaped, and the two collectively forms a funnel-form space Structure, the basal diameter of rounding you is consistent with the internal diameter of Filter column 21, and top surface diameter is consistent with the diameter of split tunnel 211, should Plant the aggregation and guiding that are provided with beneficial to reaction solution of structure so that reaction solution separation is more fully more complete.Therefore, the embodiment In the single-stranded separators of DNA on the basis of embodiment 2 cause DNA it is single-stranded be separated by filtration more thoroughly, separative efficiency is significantly carried It is high.

Embodiment 5

The present embodiment is differed only in embodiment 4, and filter membrane 22 can be placed in one any bit perpendicular to split tunnel 211 Put, two Filter columns 21 are hollow dissymmetrical structure, the diameter of filter membrane 22 is slightly larger than split tunnel 211, and the both sides of filter membrane 22 are set There is squeeze-film mechanism (not shown), displacement can not be carried out to fix filter membrane 22.

During design of primers, the affine connector mark that biotin-avidin is combined also may be selected, it is any to carry out DNA The connector of mark is used equally for being fixed on film, will not be described here.

As used the affine connector with Avidin, then the membrane system of the single-stranded separation of DNA can also select to inhale with affine Attached membrane system, it is single-stranded for separating DNA.Membrane system with affine suction-operated include silicon fiml matrix membrane, Magnetic Granular Films, it is cloudy from Sub- exchange material film, nitrocellulose filter or PA membrane, and through modification, coated magnetic bead and/or silicon dioxide film etc., This is not limited.

Double-stranded DNA separation in the present invention can also use Regular mode of the prior art, the single-stranded separation of such as DNA Kit etc., as long as can play the single-stranded structures separated and collected of DNA and method all should include within protection scope of the present invention, It will not be described here.

Embodiment 6

The present embodiment is differed only in embodiment 1：The end face of the sample needle 41 is plane, a diameter of 1.5mm, table Face finish is Ra 3.2, and shifting of the sample needle 41 described in step a described in loading methods when leaving the dNTP reagent solutions Dynamic speed is 50cm/s, and translational speed when sample needle 41 described in the step b leaves the sequencing reaction liquid is 0.4cm/ S, sequencing reaction liquid is left after being moved up and down 5 times in sequencing reaction liquid.

Embodiment 7

The present embodiment is differed only in embodiment 1：The end face of the sample needle 41 be cone, a diameter of 1.6mm, 60 degree of taper, surface smoothness is Ra 9.8, and sample needle 41 described in step a described in loading methods leaves the dNTP examinations Translational speed during agent liquid is 5cm/s, and sample needle 41 described in the step b leaves the translational speed during sequencing reaction liquid For 4.5cm/s, sequencing reaction liquid is left after being moved up and down 12 times in sequencing reaction liquid.

Finally be necessary described herein be：Above example is served only for making further detailed to technical scheme Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to the above of the invention Some the nonessential modifications and adaptations made belong to protection scope of the present invention.Finally be necessary described herein be：With Upper embodiment is served only for being described in more detail technical scheme, it is impossible to be interpreted as to the scope of the present invention Limitation, those skilled in the art belongs to according to some nonessential modifications and adaptations for making of the above of the present invention Protection scope of the present invention.

Claims

1. a kind of DNA sequencing device based on pyrosequencing, it is characterised in that including sample area, reaction zone and detection zone；

The sample area includes rotatable separator disk (2), and the separator disk (2) is located at the top of reaction zone, the separator disk (2) at least one DNA Disengagement zone is included, the Disengagement zone includes the internal hollow Filter column (21) for being provided with filter membrane (22), even There is the DNA of affine connector single-stranded and separated after being not connected with the single-stranded filterings through the filter membrane (22) of the DNA of affine connector；

The reaction zone include sample-adding portion and sample cell (3), the sample-adding portion include dNTP reagent troughs (7), sample-adding punch block (4) and The multiple sample needles (41) being mounted thereon, the separator disk (2) is located above the side of reagent trough (7)；The sample-adding punch block (4) On sample cell (3)；The sample cell (3) is provided with multiple grooves position (31)；

The sample-adding punch block (4) is disc, and the sample-adding punch block (4) can be along reagent trough (7) and sample cell (3) by move portion Between move reciprocatingly；The sample-adding punch block (4) is rotated by rotating shaft (42)；

The reagent trough (7) is provided with multiple reaction positions (74), and the side-lower of the reagent trough (7) is provided with substrate supply unit (73), The substrate supply unit (73) conveys dNTP dATP, dCTP, dGTP, dTTP tetra- by conveyance conduit (1) to reaction position (74) Plant nucleic acid primer；

The detection zone includes bioluminescent detection device (5), and the groove position (31) is connected with bioluminescent detection device (5)；

The sample area, reaction zone and detection zone are arranged in analysis station (6), and the side of the analysis station (6), which is provided with, to be used to grasp The manipulator of work.

2. the DNA sequencing device according to claim 1 based on pyrosequencing, it is characterised in that the separator disk (2) Provided with multiple collecting pipes (23), the Filter column (21) is installed in collecting pipe (23), and the filter membrane (22) is located at Filter column (21) end of one end or the non-end of Filter column (21).

3. the DNA sequencing device according to claim 2 based on pyrosequencing, it is characterised in that the collecting pipe (23) provided with dismountable upper lid (231), the upper lid (231) is connected by connect band (232) with collecting pipe (23).

4. the DNA sequencing device according to claim 1 based on pyrosequencing, it is characterised in that the move portion bag Include and the slide rail (62) above analysis station (6) and the sliding block of described sample-adding punch block (4) bottom are fixed on by mounting bracket (61).

5. the DNA sequencing device according to claim 1 based on pyrosequencing, it is characterised in that the sample needle (41) it is solid needle, the sample needle (41), which is fixed on, to be loaded in the through hole of punch block (4).

6. the DNA sequencing device according to claim 1 based on pyrosequencing, it is characterised in that groove position (31) is along sample The center of circle of product groove (3) is evenly distributed in a series of concentric circles, and the groove position (31) is made up of transparent material, and the groove position (31) is prolonged Stretch into bioluminescent detection device (5).

7. the DNA sequencing device according to claim 1 based on pyrosequencing, it is characterised in that the bioluminescence Detection means (5) includes set casing (51) and camera, and the camera is rotatable to be located in set casing (51), and the camera is located at All groove positions (31) are enclosed on the axis set.

8. a kind of DNA sequencing system based on pyrosequencing, it is characterised in that including the sequencing dress described in any of the above-described Put, and for controlling the control zone of sequencing device；The control zone includes microcomputer, Optimizing Control System, position control System, environmental control system, activity monitor system and driving control system；The environmental control system include to solution concentration and The control of pH value.