CN106591107A

CN106591107A - Sample adding device for pyrosequencing

Info

Publication number: CN106591107A
Application number: CN201710061937.3A
Authority: CN
Inventors: 刘丹; 孙悦; 陈立波
Original assignee: Wuhan Feisite Biotechnology Co ltd
Current assignee: Fist Shanghai Biotechnology Co ltd
Priority date: 2017-01-12
Filing date: 2017-01-27
Publication date: 2017-04-26
Anticipated expiration: 2037-01-27
Also published as: CN106754343A; CN107299054B; CN107299054A; CN107312710B; CN106754313A; CN106754292B; CN106754343B; CN106754292A; CN107312710A; CN106591107B

Abstract

The invention provides a sample adding device for pyrosequencing, which comprises a sample area and a reaction area; the sample area comprises a rotatable separation disc, the separation disc comprises at least one DNA separation area, the separation area comprises a hollow filter column with a filter membrane inside, and a DNA single chain connected with an affinity connector and a DNA single chain not connected with the affinity connector are separated after being filtered by the filter membrane; the reaction area comprises a sample adding part and a sample groove, wherein the sample adding part comprises a dNTP reagent groove, a sample adding needle frame and a plurality of sample adding needles arranged on the sample adding needle frame; the sample adding needle frame is positioned on the sample groove; the sample groove is provided with a plurality of groove positions. The sample adding device and the system for pyrosequencing have the advantages of simple and convenient operation and rapid detection.

Description

The sample adding device of pyrosequencing

Technical field

The present invention relates to pyrosequencing sample adding device, is specifically related to a kind of pyrophosphoric acid based on pyrosequencing and surveys Sequence sample adding device and system, belong 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 that the pyrophosphoric acid (PPi) of release is detected, pyrosequencing are 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.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 which is former by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems 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) synergism, by primer The polymerization of upper each dNTP of DNA is coupled with the release of fluorescence signal, by the release and the intensity that detect fluorescence, reaches reality When determine DNA sequence purpose (horse Yongping etc., pyrosequencing techniques and its application [J] in biology field. it is external Medical Molecular Biology fascicle 2003,25 (2):115-117).

The reaction system of liquid phase pyrosequencing is made up of reaction substrate, single-stranded, specificity sequencing primer to be measured and enzyme, instead Substrate is answered for 5 '-phosphosulfate (APS) and fluorescein (1uciferin).Liquid phase pyrosequencing course of reaction is one to anti- 4 kinds of dNTP are added to participate in the process of reaction in answering system in turn, only a kind of dNTP of often wheel reaction is participated in.If the dNTP for adding Can just with the next base pairing of DNA profiling, then its 3 ' last of 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, the PPi and APS of generation combine shape Into ATP；In the presence of luciferase, the ATP of generation is again and fluorescein combines to form oxyluciferin, while generation is visible Light.If this dNTP for adding can just with the individual identical Mismatchings of continuous n below DNA profiling, according to reaction equation Understand, n times when the visual intensity for discharging should be only 1 Mismatching, namely the light intensity discharged in course of reaction Relation proportional to the base number for matching.If the next base of the dNTP for adding and DNA profiling is mismatched, above-mentioned Reaction will not occur, and also not have the release of 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 to digital signal, Jing PC Software processes can obtain a special detection peak, and the height of peak value should be with the proportional relation of base number for matching.

After last round of reaction terminates, another kind dNTP is added, repeats above-mentioned reaction.Finally can be according to the light for obtaining The DNA sequence information to be measured that intensity peak figure can read.

It should be noted that：Catabolite deoxidation Guanosine 5'-Monophosphate (dAMP) of dATP is the inhibitor of luciferase, with The carrying out 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 sequencing sequencing length shorter (usually 20bp～30bp) sequencing technologies:Methods and goals, Nat.Rev Genet., 2004,5 (5):335-44).

Solid phase pyrosequencing is that, by 3 kinds of enzymatic chemiluminescence reactions, compared with liquid phase pyrosequencing, do not have three Adenosine phosphate bisphosphatase is participated in.Solid phase pyrosequencing course of reaction is as follows：The DNA profiling for combining primer is fixed in Support on thing and holding position is constant during the course of the reaction；After adding a kind of dNTP, in archaeal dna polymerase, ATP sulfurylases and fluorescence React under plain enzyme synergism, except no degradation reaction occurs, other reactions are identical with liquid phase pyrosequencing； Under addition, before a kind of dNTP, there is a rinsing step (washing step) to wash away upper wheel reaction residue completely, 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 be more effectively utilized by archaeal dna polymerase than dATP, it is more beneficial for reading rich in T Region.DATP α S are the mixture of two kinds of isomer Sp-dATP α S and Rp-dATP α S, 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 The concentration of S is also increasing.DATP α S can still be produced the mortifier of luciferase after Apyrase degradeds, thus dATP α S's plus Enter reading Process capabi l i ty 32.

Gharizadeh et al. is improved to 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, makes the sequencing length of pyrosequencing method increase to 50bp～100bp, the increase that length is sequenced 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 held in 2000 and analysis meeting (12th International Genome Sequencing and Analysis Conference) on, Ronaghi et al. proposes one kind and removes suppression product, reduces Sequencing length is increased to 200bp by the method for dilution effect.

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

2nd, the application progress of pyrosequencing techniques

1 application in single nucleotide polymorphism research

Single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNPs) is for occurring in recent years Three generations's genetic marker, it refers to there are 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 labellings 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 susceptibility of the colony to disease, the hereditism's base to medicine and envirment factor differential responses Plinth.

The research of SNP 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, it is found that SNP is the first step of SNP researchs, and SNP functions are ground Study carefully the purpose for being only SNP researchs.Sanger sequencing technologies have become the mainstream technology of extensive, accurate, quick discovery SNP.And Sequence verification analysis and frequency analyses are carried out to existing SNP in data base, the pyrosequencing of short sequence and checking is good at Technology is to select well, carries out SNP researchs using pyrosequencing techniques, can more save the time and reduce consuming.

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

2 applications 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.The pyrosequencing techniques such as Monstein successfully detect the V1 of helicobacter pylori 16S rRNA genetically labile With V3 region sequences, it was demonstrated that the technology can meet Rapid identification and typing to analysis of clinic pathogenic microorganism specimen.Unnerstad etc. is utilized should Technology has carried out typing 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.This technology such as Gharizadeh is to 67 Individual human papillomavirus' sample has carried out identification and typing, it was demonstrated that the technology is also very suitable for the extensive mirror of the pathogen such as HPV Fixed, typing and the research of mutation.Cheng Shaohui etc. extracts viral RNA from the Vero-6 cells of infection people's SARS virus, using Jiao Phosphoric acid sequencing technologies are sequenced to multiple base mutation sites and mutation frequency analysis.Mutation is likely to occur by sequencing analysis are multiple Site, it is determined that the virus be Beijing epidemic strain.

3 applications 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 the CYPl7 genotype of CC The probability for suffering from carcinoma of prostate for the CYPl7 genotype African Americans of TT wants height, it was demonstrated that the base in this kind of crowd is C's CYPl7 gene pleiomorphisms are in close relations with the sickness rate of carcinoma of prostate, are high-risk group.Numerous clues show, positioned at chromosome There is important relation with schizoid morbidity in the COMT genes of 22q11, and the research work of scientist could not be proposed always Strong evidence；Shifman etc. proposes a kind of effective method, and they utilize pyrosequencing techniques, the moral to 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 gene analysiss research of other diseases.

4 applications in forensic identification

With Sanger sequencing to mitochondrial DNA (mtDNA) analysis of variance, it is impossible to realize to by containing pollutant, multiple The precise quantification analysis of the mtDNA mixture of the compositions such as individual DNA, and Andreasson etc. proposes one kind and mixes for mtDNA The quantization method of the novelty 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, carry out the detection that length is 20bp, by ginseng on the gene segment of 149bp length Examine database sequence, it becomes possible to be able adequately determines origin biology of object, such as, can determine that one piece of skin histology is actually With missing crew or animal.

3rd, pyrophosphoric acid sample adding device and sequencing device and system and development

The application of pyrosequencing techniques depends on pyrophosphoric acid sample adding device and sequencing device and systematic research and exploitation. No matter which kind of pyrophosphoric acid sample adding device and sequencing device and system, its primary structure should all include two parts：Reactor part With faint light detecting portion.Reactor provides the place that carries out of reaction, faint light detecting portion be responsible for detecting reaction sends can See light.In research and application process of the people to pyrosequencing techniques, the reactor of design and use can be largely classified into 3 Class：Micro plate reactor, micro-fluidic chip reactor and micro-array chip reactor.

And business-like pyrosequencing instrument has been emerged abroad, but the report of domestic pertinent instruments research 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 of the said firm's calendar year 2001 release, system can carry out 96 tunnels simultaneously or the independent of 384 road DNA samples is sequenced, and work as survey During the of length no more than 300bp of sequence, accuracy and reliability reached 99%, with high pass at 45 minutes 1 hour the general time used Amount, quick, economic advantage.PSQ96 systems are had been widely used in the middle of basic medical research and clinical molecular diagnosis.

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

Domestic instrument research at present is at the early-stage, and the road of production domesticization is very long, in the face of the problem of every aspect, in balance After various hardware conditions required for sequencing system, it is found that the problem for primarily facing and challenge are the developments of micro sample-adding subsystem Problem.

4th, in pyrosequencing sample adding system importance

Pyrosequencing techniques and products thereof are big flux, low cost, it is more in good time, quickly and intuitively to carry out mononucleotide State property is studied and Clinical Laboratory 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 are adopted by increasing research worker, 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 Instrument such as PSQ96, GS20 are expensive for product pyrosequencing；(2) business-like pyrosequencing service latency length and It is very inconvenient；(3) although some laboratorys are in the research for carrying out the devices such as pyrosequencing chip at present, some laboratorys There is the pyrosequencing assay device of homemade, simple structure, but it is domestic without towards low side, low-cost, 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 at 50 μ L, required reaction bottom The amount of the reagents such as thing, DNA profiling and Deoxydization nucleotide 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 rapidly big, thus result in template concentrations decline It is too fast.Due to the non-linear increase of response delay that diffusion is produced, the fluorescent signals for obtaining extend on a timeline, by force Degree is reduced on the vertical scale, ultimately results in the be sequenced length of serious curtailment nucleic acid.It is generally acknowledged that caused due to follow-up sample-adding If reaction system increases within 10%, the impact to experimental result is within the acceptable range.If will be to one section The DNA segment of 20bp length is sequenced, then in the reaction system of 50 μ L, it is allowed to single sampling amount can only be less than 0.3μL。

Additionally, except being loaded precision, sample-adding interlude accuracy is similarly important.Only add in constant duration Enter the dNTP needed for single cycle, can just make the palliating degradation degree of each residual dNTP suitable, then the shadow caused to lower secondary response Sound also will be equal.There is provided benchmark could to the signal in each cycle Deng the time period, be easy to follow-up according to fluorescence signal intensity calculating Nucleotide binding number purpose automated analysiss.

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 96 orifice plate of standard can be carried out, shaken Swing, cleaning, but this set system is due to the restriction of nozzle processing technique, is loaded that micro- precision is minimum to only have 1 μ L, it is impossible to meet The nL ranks that pyrosequencing is required.Through investigation, be limited to domestic application and manufacture level, the sample adding device of production domesticization all without Method is met in pyrosequencing to sample-adding amount and the high request of repeatable accuracy.

Southeast China University Ge Jian emblems etc. disclose a kind of with faint light detection module and micro dNTP sample-adding modules as crucial mould The liquid phase pyrophosphoric acid sample adding device of block and sequencing device and system, it is disclosed that an air pressure controls micro dNTP is loaded module, It is capable of achieving to be loaded while 96 road dNTP solution, minimum sample-adding amount is 1.2 μ L, and maximum error is 13%；But signal noise is larger, Still need to a more step and improve (Ge Jianhui etc., based on the development of the gene assaying device of pyrosequencing, Southeast China University, master's degree Paper, 2006).

Wang Chunlin etc. discloses micro sample-adding system in a kind of pyrophosphoric acid nucleic acid sequencing instrument of employing piezoelectric ceramics shower nozzle, should 96 hole on-gauge plate samples can be carried out with the sample-adding in turn of 4 kinds of dNTP reagents respectively under step motor drive, sample-adding repeats essence More than 95%, single sampling minimum can reach 0.l μ L to degree.It is micro- used by above-mentioned two class preferably pyrophosphoric acid nucleic acid sequencing instrument Amount sample adding system structure is more complicated, and sample needle easily blocks up, dNTP by different modes spray in sequencing reaction liquid not with Reactant liquor contact cause it is incomplete with the reaction of reactant liquor undercompounding, it is also higher to dNTP required amounts and data are easily inaccurate； Additionally, dismounting trouble, high cost and apply under being unfavorable for specific condition.

Pyrosequencing (preosequencing) technology is a kind of new DNA sequence analysis skill developed in recent years Art, it passes through nucleotide and template combines the pyrophosphoric acid initiation enzyme cascade of rear release, promotes fluorescein luminescence and is examined Survey.It is a preferable genetic analyses technology platform, can have both carried 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 the 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 luciferase (luciferase) and the same reaction system of 4 kinds of enzyme catalysiss of bisphosphatase (apyrase) Cascade chemiluminescence reaction, reaction substrate is 5 '-phosphosulfate (5 ' phosphosulfate of adenosine, APS) and fluorescence Element.Reaction system is also including DNA to be sequenced is single-stranded and sequencing primer.In each wheel sequencing reaction, a kind of dNTP is added, if should DNTP and template are matched, and during polymerase just can be incorporated into primer strand and discharge the pyrophosphoric acid group of equimolar number (PPi).Sulfurylase is catalyzed ASP and PPi and forms ATP, and the latter drives the fluorescein of luciferase mediation to oxyluciferin Conversion, sends the visible light signal being directly proportional to ATP amounts, and by Pyrogram^TMBe converted into a peak value, its highly with reaction in The nucleotide number of incorporation is directly proportional.According to add dNTP types and fluorescence signal intensity can real time record template DNA core Nucleotide sequence.Replace adenosine triphosphate (dATP) with effectively with the adenosine triphosphate (dATP α S) of α-sulfuration in experimentation Utilized by archaeal dna polymerase, and do not recognized by luciferin.As 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 purification Spisomer The use dATP α S catabolites of dATPaS suppress this problem of bisphosphatase activity preferably to be solved so that sequencing length Up to 10bp, range of 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 as to To determine DNA sequence.By containing by the reactant liquor of 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 moves to other reactive tank, carries out luminescence-producing reaction, and passes through during reactant liquor is moved The region of the enzyme for decomposing remaining nucleic acid primer is fixed with, after remaining nucleic acid primer is decomposed, makes pyrophosphoric acid be converted into ATP, Import chemiluminescence reaction groove.It is but the drawbacks of prior art is to must be added to substantial amounts of substrate and enzyme is reacted, anti-to ensure Should carry out completely, clean after needing afterwards to react away unnecessary substrate again, this not only adds course of reaction, increase every The cleaning of one step and reaction difficulty, can also cause very serious waste to reagents such as substrates, and the response time is long, waste of manpower thing Power, virtually reduces pyrosequencing popularization in the market and uses potentiality.

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

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

The single-stranded isolation technics of DNA are one of modal 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 The field such as animal husbandry, food and health, the energy and chemical industry, environmental monitoring and medical diagnosiss and detection.Additionally, DNA single-stranded suction It is attached, extract with isolation technics water quality, water source, biomaterial, biological fluid (as blood, serum, blood plasma, cerebrospinal fluid, urine, Tear, perspiration, Digestive system, seminal fluid, juice, tissue fluid, vomitus, feces), tissue/cell and microbial lytic liquid, difference The analysis detection of biology, chemical molecular and the medicines such as the albumen in source, nucleic acid etc., separate and purification and oligonucleotide, many The aspects such as the synthesis of peptide, lead compound and medicine are widely used, closely bound up with daily life, in biological medicine Field has very important status.

In biomedicine field, the deficiency of the conventional single-stranded separation methods of DNA presence is as follows：

1. thermal denaturation or alkali process.Double stranded PCR products are mainly heated or alkali process by this kind of method, due to DNA Double-strand hydrogen bond under high temperature or a certain degree of alkaline environment can rupture 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 purification not as single stranded DNA, is used for The double-strand of DNA is separated.

2.T7 reverse transcription methods.This kind of method is, plus T7 promoteres, to be produced with purification PCR amplifications at an end of PCR primer 5 ' 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 the single-stranded purity of DNA for obtaining It is higher, but whole separation process need to be divided into two big steps and complete, operation inconvenience, and the time is longer, and need to strictly control RNase Pollution, therefore with certain limitation.

3. Exonucleolytic enzyme process (Higuchi and Ochman, Nucleic.Acids Res., 1989,17:5865).By It is phosphorylated in a PCR primer, PCR primer when exonuclease digestion is used, do not cut by the primer amplification chain being phosphorylated Cut, after digestion, enzyme is heated inactivation.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, by 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, under the conditions of partial denaturation, heterologous Because of the presence that has mispairing district and more changeableness, retention time in the chromatography column is shorter than homoduplex to double-strand, therefore is first eluted down Come, the elution curve of bimodal or multimodal is shown as in chromatogram.As a PCR primer is by biotin labeling, its PCR is expanded Chain in DHPLC, can and another common chain separate (Dickman and Hornby, Anal.Biochem., 2000,284: 164-167).DNA needed for the method directly can be obtained from double stranded PCR products in 15min is single-stranded, but this kind of method Implement to need supporting sufficiently expensive instrument, therefore be difficult to all the time popularize.

5. magnetic capture method.The surface of superparamagnetic nano particle is improved with nanotechnology and surface modification Afterwards, it is prepared into superparamagnetism silicon oxide nanometer magnetic bead.The magnetic bead can specifically recognize with nucleic acid molecules on micro interface and Efficiently combine.Using the superparamagnetism of silicon oxide Nano microsphere, in Chaotropic salt (guanidine hydrochloride, guanidinium isothiocyanate etc.) and outward Plus in the presence of magnetic field, can separate from the DNA in blood, animal tissue, food, pathogenic microorganism equal samples and RNA, so Target is obtained with NaOH process afterwards single-stranded.This kind of method is simple to operate, the used time is short, and whole flow process of extracting is divided into four steps, mostly may be used To complete within 36-40 minutes, and safety non-toxic, the toxic reagents such as benzene in traditional method, chloroform are not used, 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 causes the DNA for extracting single-stranded pure Degree is high, concentration is big, but the coating magnetic bead used in this kind of method is costly, and needs to separate by magnetic frame, not only separates It is relatively costly, it is also inconvenient, therefore the popularization of the technology is limited to a certain extent.

6. asymmetric PCR.Above method is both needed to extra process is carried out after PCR, and asymmetric PCR can be expanded in PCR While prepare DNA it is single-stranded.Primer of the conventional asymmetric PCR using two inequalities, is normally expanded in the circulation for starting Increase.With the increase of circulation, measure few primer and gradually exhausted, and the primer of excess can continue straight line amplification to generate DNA single-stranded (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 higher, but the ratio of its primer needs optimization, and non-spy The chance of different amplification increases, additionally, the single-stranded separation processes of DNA need to rely on electrophoresis, separable programming is complicated, and electrophoresis is often visible Smear, which takes and is not obvious.

Above-mentioned several separation methods are respectively provided with certain limitation, therefore, it is single-stranded to DNA detached operable in order to meet Property and cost-effectiveness requirement, DNA of the prior art is single-stranded to use integrated extraction work station, will carry streptavidin Affine connector is combined with DNA double chain, and work station has sucking filtration pin and supporting pump, and the DNA with reference to after is affine, and connector passes through Sucking filtration adsorbs the filter membrane bottom in sucking filtration pin, and work station is furnished with track and related system, and sucking filtration pin is moved to Sheng after terminating by sucking filtration In having the disk of NaOH, by alkali process solution Double helix, it is single-stranded to obtain DNA, and after sucking filtration, cleaning is collected again.Sucking filtration pin is general 24 (4*6) it is one group, during use, must assure that the sample or reagent of q.s is to ensure the normal operation of work station therefore this DNA single-stranded collection mode is very dumb, can only with fixed amount add work station be operated, and it is substantial amounts of be lost in it is many Produce in secondary sucking filtration and transfer process, this is very unfavorable to micro-collection, and sucking filtration pin group needs to be operated simultaneously, this All there is certain volume requirement to each part of work station, whole work station takes up room very big.Huge system is caused in DNA In single-stranded separation operation process, micro- detached dowel needs liquid relief back and forth, and operation is very loaded down with trivial details, not only separation cycle length, efficiency It is low, and integral device is expensive, causes DNA single-stranded detached costly, in addition it is also necessary to expend substantial amounts of reagent and other resources, It is extremely uneconomical.Additionally, sucking filtration pin is metal material in the work station, and it is expensive, re-use after often processing, therefore easily Cause the cross-contamination between residue, reliability is not high, certain doing can be caused to the accuracy of separation and testing result Disturb and affect.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- Detached dowel adsorbs, and causes the DNA proportion of single chain for obtaining to reduce, 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

For the problems referred to above that prior art is present, it is an object of the invention to provide a kind of easy to operate, quick detection Pyrosequencing sample adding device and system.

For achieving the above object, the technical solution used in the present invention is as follows：

Pyrosequencing sample adding device, including sample area and reaction 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 that inside is provided with the hollow Filter column of filter membrane, is connected with affine connector Separate after the single-stranded membrane filtrations described in of DNA of the single-stranded and not connected affine connectors of DNA；

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 are 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 groove positions.

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 detached dowel bottom, that is to say, that the single stranded DNA after separation is on filter membrane.

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

Another kind of preferred mode, the separator disk are provided with multiple collecting pipes, and the Filter column is installed in collecting pipe, institute Filter membrane is stated positioned at the non-end of 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 with collecting pipe by connect band.

Single stranded DNA after separation to be connected with the single-stranded of affinity body, needs the chain wash to filter membrane on filter membrane It is de-, containing another complementary strand without affine connector in filtrate, the single stranded DNA in filtrate can be entered when needing backward sequencing Row is collected.When needing to collect a chain of the band affine connector on filter membrane, collecting pipe can adopt the collecting pipe for reclaiming, Now collecting pipe only plays a part of Recycling of waste liquid, and recovery uses 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 tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP of dNTP 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 above analysis station and the sample-adding punch block by installing rack The slide block of bottom.

Preferably, the rotating shaft is provided with motor.

Preferably, the sample needle be solid needle, the sample needle be fixed on sample-adding punch block through hole in.

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

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

It is a further object of the present invention to provide a kind of pyrosequencing device, including any of the above-described sample adding device is with unification Detection zone is formed, and the detection zone includes bioluminescent detection device, and the groove position is connected with bioluminescent detection device.

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

The present invention also aims to another kind of pyrosequencing system is provided, including above-mentioned sequencing device, and For controlling the control zone of sequencing device；The control zone includes microcomputer, Optimizing Control System, position control system, ring Border control system, activity monitor system and driving control system；The environmental control system is included to solution concentration and pH value Control.

For the problems referred to above that prior art is present, the advantage of pyrosequencing sample adding device of the present invention is：

(1) by it is DNA single-stranded by way of filter centrifugation being extracted, the economization for realizing the collection of a sample one is little Typeization is collected, there is provided a kind of single-stranded separation method of rapid DNA and device of efficiently low loss.This method is simple to operation, obtains Target sample time short efficiency high is taken, be can be used for the single-stranded collection of trace amount DNA and extracted, sample loss can almost be ignored, make It is low with the few requirement to equipment of reagent, in separation process without the need for suction pump, device configuration is enormously simplify, reduce equipment total Cost, is effectively simplified operating procedure, shortens the operating time, reduces working strength, improves work efficiency；Using with it is normal The supporting Filter column of rule centrifuge tube not only ensure that detached quality and efficiency, also so that separation process is simply easily realized, filter The filter membrane of post is separated by physics mode is single-stranded to DNA, reduces detached difficulty and condition is required.Filter membrane is by homogenizing Material with identical structure composition, can two-way exchange use, increase the designability of Filter column, one end not only can be made as Hollow structure with filter membrane, enriches the structure and species of the single-stranded segregation apparatuss of DNA, on the premise of identical function is guaranteed, increases The application occasion of the DNA single-stranded segregation apparatuss 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 adopted, can be exchanged up and down and be used cooperatively.The single-stranded segregation apparatuss of this DNA are moulded using cheap PC Material, economic and practical, the design of infundibulate inner chamber are conducive to the aggregation and guiding of reactant liquor so that reactant liquor separates 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.Additionally, this DNA lists Chain separation device applies also for commercially available centrifuge tube, standardized designs so that the single-stranded segregation apparatuss versatilities of this DNA are extremely strong, is suitable for Face is extremely wide, therefore its application prospect is very wide.

(2) present invention is capable of achieving the micro to sample of dNTP reagents using the sample needle of a simple structure, has abandoned transmission Hollow needle tube take out spray formula to sample loading mode, only by the sample needle of the sample needle from the absorption affinity between liquid and its different The control of the translational speed difference in liquid is capable of achieving microsampling sample-adding, and only by sample needle in the sequencing reaction liquid on Lower motion is capable of achieving stirring for several times and causes the more complete result of enzyme reaction accurately, and this loading methods and its device are suitable for any In detection means, dismounting is easy, cleans simple and convenient, and various sample adding devices of the prior art such as stifled pin will not occur not Foot, sample-adding repeatable accuracy are 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) pyrosequencing sample adding device of the invention, manipulates whole process using mechanical hand, without infection, accuracy rate Height, improves know clearly efficiency and precision.

In a word, the pyrosequencing sample adding device and system that the present invention is provided reduces the consumption of substrate and enzyme system, inspection Survey accurate, response speed is fast, integrated height, and can detect one or more SNP site and Single-stranded DNA fragments, enzyme system simultaneously And substrate has broken the monopolization of part producing business, price is greatly reduced, and sample adding device and sequencing device and system structure are accurate, Consumption requirement to DNA fragmentation to be measured and substrate is low, reduces the testing cost of pyrosequencing, expands pyrosequencing Use range.

Description of the drawings

Fig. 1 is the structural representation of the pyrosequencing sample adding device of the present invention.

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

Fig. 3 is that of the DNA Disengagement zone provided by the present invention for pyrophosphoric acid uses preferred embodiment.

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

Fig. 9 is the sequence spectrum that measured using the sample adding device provided by the present invention for pyrosequencing system 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 dress Put；51st, set casing；6th, analysis station；61st, installing rack；62nd, slide rail；7th, reagent trough；71st, dry slot；72nd, rinse bath；73rd, substrate Supply unit；74th, react position.

Specific embodiment

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

Embodiment 1

Fig. 1 to Fig. 3 shows the first embodiment of pyrosequencing sample adding device of the present invention.Pyrophosphoric acid of the present invention Sequencing sample adding device, can be used for pyrosequencing detection and analysis DNA sequence, and DNA sequence to be sequenced is target sequence, by target DNA sequence carries out pyrosequencing after being expanded.

The pyrosequencing sample adding device of the present embodiment includes DNA sequencing device and control zone, and 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, reaction zone and inspection Area is surveyed by control zone monitoring, control.Whole analysis process adopts Robot actions.

Before carrying out pyrosequencing, need first to expand the DNA profiling of band sequencing, to reach what amplification was required DNA concentration.When amplimer is designed, affine connector on amplimer, is carried, 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 need single stranded DNA, separator disk 2 to include at least one DNA Disengagement zone, and DNA Disengagement zone adopts physical filtering Mode, Disengagement zone includes that inside is provided with the hollow Filter column 211 of filter membrane 22, and filter membrane 22 is high molecular nano-microsphere structure, As the aperture between high molecular nano-microsphere is less than the diameter of affine connector, will be the DNA with affine connection body tag 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 In the end of one end of Filter column 21.The external diameter of 21 hypomere of Filter column is identical with the internal diameter of collecting pipe 23.

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

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

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

As shown in figure 4, in order to the filter membrane 22 in the present embodiment is not moved in piping and druming or centrifugal process, the present embodiment In further preferably film pressing device is provided with above filter membrane 22, film pressing device includes pad and/or press mold frame.The liquid of purification to be separated Body is contacted with filter membrane 22 after first passing through pad, and pad is preferably fibrous material, can tolerate soda acid and most of organic solvent, 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, compresses filter membrane 22 by mechanical pressure, makes filter membrane 22 not occur to move during the use such as piping and druming or centrifugation It is dynamic, cause to collect loss.

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, the multiple sample needles 411 for being loaded punch block 4 and 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, is loaded punch block 4 and can be moved reciprocatingly between reagent trough 7 and sample cell 3 by move portion.Move portion Including the slide block of 4 bottom of slide rail 62 and sample-adding punch block being fixed on by installing rack 61 above analysis station 6.Dry slot 71 is true Empty dry section.

Reagent trough 7 is provided with four reaction positions 74, and the side-lower of reagent trough 7 is provided with substrate supply unit 73, substrate supply unit 73 The different nucleic acid primers of dNTP are conveyed by conveyance conduit 1 to reaction position 74.Reaction position 74 to be measured is used to hold DNA to be sequenced Sequence, substrate supply unit provide dNTP, with target DNA hybridization, provide environmental basis for hybridization.For pyrosequencing DNTP include tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP, substrate for hybridizing with target DNA, and need reaction Add related enzyme systems catalytic reaction to carry out in system, specially archaeal dna polymerase, carry out complementary strand synthesis reaction, close in complementary strand Into when the by-product pyrophosphoric acid that obtains change into ATP, ATP and luciferin reaction are made in the presence of luciferase, are lighted.Such as There is complementary strand synthesis in fruit, then produce pyrophosphoric acid, as a result light, therefore by monitoring to which, can learn that generation is complementary Chain synthesizes, i.e., the base species that group enters thereby determines that DNA sequence.

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 the DNA single-stranded requirement according to reaction system It is transferred in reaction position 74, after adding enzyme system, sequentially adds dNTP, 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 is used for detection and judges whether the site is reacted and is lighted, and then sentences Break and the base sequence in the site.

Sample-adding punch block 4 is located on sample cell 3；Sample cell 3 is provided with multiple groove positions 31, has sequencing reaction liquid in groove position 31. Groove position 31 is evenly distributed in a series of concentric circulars along the center of circle of sample cell 3, and groove position 31 is made up of transparent material, and groove position 31 extends into Bioluminescent detection device 5.Bioluminescent detection device 5 includes set casing 51 and camera, and camera is rotatable positioned at set casing In 51, camera is located on the axis that all groove positions 31 are gathered.Camera is commercially available iKon-M deep refrigeratings image CCD camera, and Connect computer and the spectrogram of 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), wherein 41 a diameter of 1.5mm of sample needle, 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 41 periphery of sample needle to adhere to the dNTP reagents；Step Rapid b, will adhere in the 41 insertion groove position 31 of sample needle of the dNTP reagents, then makes sample needle 41 leave 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 step b Translational speed when pin 41 leaves the sequencing reaction liquid is 1cm/s, to move up and down and leave sequencing after 8 times in sequencing reaction liquid Reactant liquor.

Sequencing spectrograms of the Fig. 9 obtained by the use pyrophosphoric acid nucleic acid sequencing detection means of the present embodiment, as shown in Figure 9, wherein Order TCATATTCGCCAGT of dNTP is sequentially added, wherein non-appearance when adding T first, and be not subsequently inconsistent with actual sequence DNTP reagent solutions also non-appearance (being marked with circle in Fig. 9, i.e. T, T, C), the sequence which measures is CAATATTCGCCAGGT, Completely the same with sample actual sequence, degree of accuracy is 100%, detects that the time of single base is less than 1.5 minutes, and 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 pyrosequencing sample adding device of the present invention can preferably transmit substrate and DNA, reduce and pass Loss during passing.

The shape of reaction zone is not limited, and the concrete structure in sample adding device and sequencing device and system can be according to the need of detection Adjustment is designed, any planform and relative position that may realize pyrosequencing is deemed to fall the present invention's Within protection domain.

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

Enzymatic mixture includes：

Substrate mixture includes：

APS 0.4mg/L；

Firefly luciferin 0.4mmol/L.

In course of reaction, the optimum pH value for enzymatic activity is selected to react in each step, it is right to need after reaction PH value in reaction system is adjusted to adapt to other reactions, the reaction condition ordinary skill in concrete reaction system Personnel can be drawn according to prior art.For example, when in washing step comprising apyrase removing excessive nucleoside When sour species and ATP, because the seriality of process step, buffer can be used together with apyrase, its pH The horizontal optimization of the clean property of apyrase can be made.Then, polymerase used in step is mixed in next nucleotide, Can be using the different buffer with the optimal pH condition for polymerase, to make the clean property optimization of polymerase.In addition, every Kind optimized buffer liquid may include the preferred counter ion counterionsl gegenions for every kind of enzyme, such as apyrase is buffered Ca for liquid²⁺With the Mg for polymerase buffer²⁺。

As shown in figure 9, in reaction system, detection sequence is CAATATTCGCCAGGT, wherein sequentially adding the order of dNTP For TCATATTCGCCAGT, wherein non-appearance when adding T first, and the dNTP reagents not subsequently being inconsistent with actual sequence are not gone out yet Peak, the sequence which measures are CAATATTCGCCAGGT, and completely the same with sample actual sequence, degree of 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 by bioluminescence other can detect that the device of bioluminescence can be using to examine Device is surveyed, here is not limited.

The pyrosequencing sample adding device based on pyrosequencing in embodiment of the present invention, also including each sample Liquid supply and passing away between storage device, liquid supply passage are used for the reagent and sample for conveying question response, liquid Passing away is expelled to collecting part for the liquid after reaction is terminated 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.

In the sample adding device and sequencing device and system of the present invention in the reaction, need to ensure that each portion is operated in optimum In the environment of carry out, enzyme system needs to be reacted in active highest reaction system, complete to ensure reaction, therefore control zone In be provided with can ensure that these reactions can some components for carrying out of effective, including centrifuge, pH meter, heating component, control The standing structure of the sequencing equipments such as signal transmission component processed.

The system and method for embodiment of the present invention may include to be carried out some designs, divided using computer-readable medium Analysis or other operations, such media storage have the instruction for performing on the computer systems.For example, process institute's detection signal And/or analysis some embodiments of the data of sequencing result system and method generation, wherein processing and analyzing 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 Become pel array.May also comprise interfacial level controller, wherein may include any different oneself know or future software program, be used for Input and output interface are provided.For example, interface may include so-called " graphic user interface (Graphical User Interfaces, commonly referred to GU I) ", which can provide the user one or more image and show.Using the common skill of association area Oneself selection for knowing of art personnel or input mode, interface usually can receive user input.

In identical or alternate embodiment, application on computers (can be led to using so-called " Command Line Interface " is included Frequently referred to CLI) at interior interface.CLI is the commonly provided based on the text applied with user's interphase interaction.Generally, order line circle Face is by display with line of text form display output receives input.For example, " order that some implementation procedures may include so-called Row interpretive program (shell) " such as person of ordinary skill in the relevant oneself Unix command line interpreter (Unix for knowing Shell), or using the Microsoft Windows Powershell of object oriented program architecture, 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 operating system may be selected any operating system of the prior art, as long as this Art personnel can be used for the work such as processing detection result and data and think to include within protection domain.

DNA sequence can be widely used for based on the pyrosequencing sample adding device of pyrosequencing in the present invention It is determined that, diagnosis, check, the determination of SNP site, diagnosis etc., be capable of achieving to be sequenced while certain sample, condition of the principle to sequencing Require low, it is not necessary to extra to increase the expensive experiment reagent such as excitation source and fluorescent agent, be capable of achieving easy cheap DNA and survey Sequence works, and testing result is stable, and accuracy rate is high, and overcoming conventional equipment fully can not be carried out or complementary strand synthesis reaction The drawbacks of first excessively carrying out, reaction volume is little, and the work of the stage of reaction can be completed within the shorter time.

The pyrosequencing sample adding device provided using the present invention and the analysis method of system, are comprised the following steps, its In be not disclosed part 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, and sepharose 4B is a diameter of 30 μm, and surface is covered with biotin and Streptavidin, amplification DNA fragmentation afterwards is spontaneously specifically bound with the coated sepharose 4B of Streptavidin；

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

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

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

(5) adjust pH value：Appropriate eluting 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, is gently blown and beaten after addition to DNA is single-stranded and is suspended completely, use after sucking-off In pyrosequencing or 4 DEG C of sealing preserves.

(7) it is loaded：Any dNTP reagent is dipped by sample needle 41 causes the attachment of 41 periphery of the sample needle 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 that realization completes sequencing, preferably by it is 4 kinds of dNTP reagents any one or more depending on sequence to be measured with which kind of During order adds 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.

Due to 22 diameter of filter membrane with diameter greater than the single-stranded segregation apparatuss of DNA of affine connector, therefore pass through DNA is single-stranded During filter membrane 22, be not associated with affine connector DNA is single-stranded and other impurities by filter membrane 22, and can combine affine connector Single-stranded being left on film and cannot pass through, this filtration is physical property.

In DNA separation processes in the present embodiment, involved solution, parameter and other separation conditions, are the present embodiment In preferred embodiment, any experiment condition, parameter and solution that can play respective action with reference to prior art can use Involved in the present invention to isolate and purify process, the design parameter and solution in the present embodiment selects to should not be used as to the present invention Restriction.

Spectrogram and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) are sequenced obtained by the present embodiment It is the concordance in the range of theoretical error with 1 the data obtained of embodiment, its sample-adding repeatable accuracy is 96%.

Embodiment 2

As 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 sets In the non-end face of Filter column 21, when needing to collect, usual manner, such as centrifugation etc. after 21 two of Filter column is exchanged, are adopted, Can elute all DNA on filter membrane 22 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 are vertically positioned in the middle part of hollow cylinder, the Filter column 21 can two ends exchange use, hollow posts The internal diameter of body is 4.5mm, 22 a diameter of 4.7mm of filter membrane, is allowed to be brought into close contact by filter membrane 22 is pressed in Filter column 21 by force Leave no gaps, 22 spacing squeeze-film mechanism (not shown) of filter membrane is added in cylinder, displacement cannot be carried out to fix filter membrane 22.By In the single-stranded segregation apparatuss of the DNA either still functionally two ends all same in structure, thus the single-stranded segregation apparatuss of the DNA without Liquid feeding end and liquid outlet need to be distinguished, a use can be arbitrarily selected at two ends when in use.

After unnecessary NaOH being washed away through step (4), exchange the two ends of Filter column 21, add collection liquid, it is static More than 1mins, can carry out elution step without the need for piping and druming, and during eluting, centrifuge speed is less than 10000rpm, is at least centrifuged 2min.DNA concentration can be detected by nanodrop after collection, carry out pyrosequencing.

Spectrogram and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) are sequenced obtained by the present embodiment It is the concordance in the range of theoretical error with 1 the data obtained of embodiment, its sample-adding repeatable accuracy is 95%.

Embodiment 3

As shown in fig. 6, the present embodiment and embodiment 1 are differed only in, the mistake of the single-stranded segregation apparatuss of DNA in embodiment 3 Filter post 21 adopts space circular platform type structure, filter membrane 22 to be arranged at the public top surface of two circular platform type Filter columns 21, and circular platform type is filtered The top surface diameter of post 21 is consistent with the diameter of filter membrane 22, and 22 both sides of filter membrane are provided with squeeze-film mechanism (not shown), with fixed filter Film 22 cannot 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 It is scheduled on the boss of collecting pipe 23.

The step of using the separation method of segregation apparatuss affiliated in the present embodiment with described in embodiment 1, is identical, with reality Apply differing only in for example 1：After unnecessary NaOH being washed away through step (4), exchange the two ends of Filter column 21, add and receive Liquid collecting, static more than 1mins can carry out elution step without the need for piping and druming, and during eluting, centrifuge speed is less than 10000rpm, extremely 2min is centrifuged less.DNA concentration can be detected by nanodrop after collection, carry out pyrosequencing.

Embodiment 4

As shown in fig. 7, differing only in for the present embodiment and embodiment 1, in order to preferably assemble and guide reactant liquor, makes Reactant liquor is separated more fully more completely, evades filtration 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 is connected with the two, two Filter columns 21 and split tunnel 211 Concentrically axis, filter membrane 22 are arranged on the plane of symmetry of split tunnel 211, and 22 both sides of filter membrane are provided with squeeze-film mechanism and (do not show in figure Go out), displacement cannot 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 It is scheduled on the boss of 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 are consistent with 21 internal diameter of Filter column, 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 reactant liquor of structure so that reactant liquor separates more fully more complete.Therefore, the embodiment In the single-stranded segregation apparatuss of DNA cause that DNA is single-stranded to be separated by filtration more thoroughly on the basis of embodiment 2, separation efficiency is significantly carried It is high.

The step of using the separation method of segregation apparatuss affiliated in the present embodiment with described in embodiment 1, is identical, with reality Apply differing only in for example 1：After unnecessary NaOH being washed away through step (4), exchange the two ends of Filter column 21, add and collect Liquid, static more than 1mins can carry out elution step without the need for piping and druming, and during eluting, centrifuge speed is less than 10000rpm, at least Centrifugation 2min.DNA concentration can be detected by nanodrop after collection, carry out pyrosequencing.

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, and slightly larger than split tunnel 211,22 both sides of filter membrane set the diameter of filter membrane 22 There is squeeze-film mechanism (not shown), displacement cannot be carried out to fix filter membrane 22.

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

As used the affine connector with Avidin, then the single-stranded detached membrane systems of DNA can also be selected with affine suction Attached membrane system, it is single-stranded for separating DNA.Include silicon fiml matrix membrane, Magnetic Granular Films, the moon with affine adsorbing membrane system Ion exchange material film, nitrocellulose filter or polyamide membrane, and modified, coated magnetic bead and/or silicon dioxide film etc., Here is not limited.

Double-stranded DNA in the present invention separates and Regular mode of the prior art, the single-stranded separation of such as DNA may also be employed Test kit etc., as long as the single-stranded structures for separating and collecting of DNA can be played and method all should be included within protection scope of the present invention, Will not be described here.

Embodiment 6

The present embodiment is differed only in embodiment 1：The end face of the sample needle 41 be plane, a diameter of 1.5mm, table Face fineness 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 step b leaves the sequencing reaction liquid is 0.4cm/ S, to move up and down in the sequencing reaction liquid and leave sequencing reaction liquid after 5 times.

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 are 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 step b leaves the translational speed during sequencing reaction liquid For 4.5cm/s, to move up and down in sequencing reaction liquid and sequencing reaction liquid is left after 12 times.

Finally be necessary described herein be：Above example is served only for making further in detail technical scheme Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art's 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 Restriction, some nonessential modifications and adaptations that those skilled in the art's the above of the invention is made belong to Protection scope of the present invention.

Claims

1. pyrosequencing sample adding device, it is characterised in that including sample area and reaction zone；

The sample area includes rotatable separator disk (2), and the separator disk (2) is positioned at the top of reaction zone, the separator disk (2) including at least one DNA Disengagement zone, the Disengagement zone includes that inside is provided with the hollow Filter column (21) of filter membrane (22), even Have the DNA of affine connector single-stranded and not connected affine connector the single-stranded filter membranes described in (22) of DNA filter after separate；

The reaction zone includes 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 above the side of reagent trough (7)；Sample-adding punch block (4) On sample cell (3)；The sample cell (3) is provided with multiple groove positions (31).

2. pyrosequencing sample adding device according to claim 1, it is characterised in that the separator disk (2) is provided with many Individual collecting pipe (23), the Filter column (21) are installed in collecting pipe (23), and the filter membrane (22) is positioned at Filter column (21) one end End or Filter column (21) non-end.

3. pyrosequencing sample adding device according to claim 2, it is characterised in that the collecting pipe (23) is provided with can The upper lid (231) of dismounting, the upper lid (231) are connected with collecting pipe (23) by connect band (232).

4. pyrosequencing sample adding device according to claim 1, it is characterised in that the reagent trough (7) is provided with many Individual reaction position (74), the side-lower of the reagent trough (7) are provided with substrate supply unit (73), and the substrate supply unit (73) is by defeated Send pipeline (1) that tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP of dNTP are conveyed to reaction position (74).

5. pyrosequencing sample adding device according to claim 1, it is characterised in that the sample-adding punch block (4) is circle Dish type, the sample-adding punch block (4) can be moved reciprocatingly between reagent trough (7) and sample cell (3) by move portion；The sample-adding Punch block (4) is rotated by rotating shaft (42).

6. pyrosequencing sample adding device according to claim 5, it is characterised in that the move portion is included by peace Shelve slide rail (62) and the slide block for being loaded punch block (4) bottom that (61) are fixed on above analysis station (6).

7. pyrosequencing sample adding device according to claim 5, it is characterised in that the sample needle (41) is solid Pin, the sample needle (41) are fixed in the through hole of sample-adding punch block (4).

8. pyrosequencing sample adding device according to claim 1, it is characterised in that groove position (31) are along sample cell (3) The center of circle in a series of concentric circulars it is evenly distributed, the groove position (31) is made up of transparent material, and the groove position (31) extends into life Thing luminescence detection apparatus (5).

9. a kind of pyrosequencing device, it is characterised in that：Including the arbitrary described sample adding device of claim 1～8 with unification Detection zone is formed, and the detection zone includes bioluminescent detection device, and the groove position is connected with bioluminescent detection device.

10. a kind of pyrosequencing system, it is characterised in that：Including the sequencing device described in claim 9 and one and it is used for The control zone of control sequencing device.