CN106591107B - Sample adding device for pyrosequencing - Google Patents

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 devices, are specifically related to a kind of pyrophosphoric acid survey based on pyrosequencing Sequence sample adding device and system belong to genetic test field.

Background technique

One, pyrosequencing overview

Pyrosequencing techniques (Pyrosequencing) grew up in 1987, are based in DNA synthesis process The sequencing technologies of pyrophosphoric acid (PPi) detection of release, pyrosequencing react the process under a series of catalytic action of enzymes It is middle to generate the visible light proportional to the aggregate number of deoxynucleotide triphosphates (dNTP), by can reach to visible detection Measure the purpose of DNA sequence dna.There are two types of implementation methods for pyrosequencing: liquid phase pyrosequencing (Liquid Phase ) and solid phase pyrosequencing (Solid Phase Pyrosequencing) Pyrosequencing.

Liquid phase pyrosequencing is the enzyme cascade chemiluminescence reaction in the same reaction system by 4 kinds of enzymatics, former Reason is: after primer and template DNA annealing, in archaeal dna polymerase (DNA polymerase), ATP sulfurylase (ATP s μ Lfurylase), under luciferase (1uciferase) and apyrase (Apyrase) synergistic effect, by primer The polymerization of each upper dNTP of DNA and the release coupling of fluorescence signal are got up, and by detecting the release and intensity of fluorescence, reach real When measurement DNA sequence dna purpose (horse Yongping etc., pyrosequencing techniques and its external in application [J] of molecular biology field Medical Molecular Biology fascicle 2003,25 (2): 115-117).

The reaction system of liquid phase pyrosequencing is made of reaction substrate, single-stranded, specific sequencing primer to be measured and enzyme, instead Answering substrate is 5 '-phosphosulfates (APS) and fluorescein (1uciferin).Liquid phase pyrosequencing reaction process is one to anti- It answers and the process that 4 kinds of dNTP participate in reaction is added in system in turn, every only a kind of dNTP of wheel reaction is participated in.If the dNTP being added Can just with next base pairing of DNA profiling, then its be added under the action of archaeal dna polymerase sequencing primer 3 ' end End, while releasing the pyrophosphoric acid (PPi) of a molecule；Under the action of ATP sulfurylase, PPi the and APS combination shape of generation At ATP；Under the action of luciferase, the ATP of generation is combined with fluorescein again and is formed oxyluciferin, while being generated visible Light.If this dNTP being added can just with a identical Mismatchings of n continuous below DNA profiling, according to reaction equation It is found that the luminous intensity discharged in n times when the visual intensity released should be only 1 Mismatching namely reaction process Relationship proportional to the base number to match.If next base of the dNTP and DNA profiling that are added mismatch, above-mentioned Reaction will not occur, the also not release of visible light.The dNTP and ATP of reaction are not participated in nucleolytic enzyme Apyrase's The lower degradation of effect.

The visible light that every wheel reaction releases is converted by Weak light detection device, then is treated as digital signal, through PC Software processing can be obtained a special detection peak, and the height of peak value should be with the proportional relationship of base number that matches.

To it is last round of after reaction, be added another kind dNTP, repeat above-mentioned reaction.It 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 is to be noted that the catabolite deoxidation Guanosine 5'-Monophosphate (dAMP) of dATP is the inhibitor of luciferase, with Reaction progress, concentration can be higher and higher, can hinder continuing for pyrosequencing chemiluminescence reaction.This is also burnt The main reason for length shorter (usually 20bp~30bp) (Shendure J, et a1.Advanced is sequenced in phosphoric acid PCR sequencing PCR 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 Adenosine phosphate bisphosphatase is participated in.Solid phase pyrosequencing reaction process is as follows: the DNA profiling for combining primer is fixed in branch It supports on object and holding position is constant during the reaction；After a kind of dNTP is added, in archaeal dna polymerase, ATP sulfurylase and fluorescence It reacts under plain enzyme synergistic effect, in addition to not having degradation reaction, other reactions are identical with liquid phase pyrosequencing； There is rinsing step (washing step) to wash away upper wheel reaction residue completely before a kind of dNTP under addition, does not have The accumulation of inhibition product.

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

Ronaghi etc. improves signal-to-noise ratio (the Ronaghi M.et of pyrosequencing using dATP α S substitution dATP 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 than dATP by archaeal dna polymerase, it is more advantageous to and reads rich in T Region.DATP α S is the mixture of two kinds of isomers Sp-dATP α S and Rp-dATP α S, and polymerase can only utilize Sp-dATP α S. Optimum response efficiency in order to obtain, it is necessary to keep the Sp-dATP α S of optium concentration in the reaction system, at the same time Rp-dATP α The concentration of S is also increasing.DATP α S can still generate 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. improves this, and pure Sp-dATP α S is only added in they in the reaction, and is added without Useless Rp-dATP α S, improves the efficiency of reaction, greatly reduces the concentration for inhibiting product, luciferase is tieed up The activity for holding the long period makes the sequencing length of pyrosequencing increase to 50bp~100bp, and 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 and analysis meeting (12th International Genome held in 2000 Sequencing and Analysis Conference) on, Ronaghi et al. proposes a kind of removal and inhibits product, reduces Sequencing length is increased to 200bp by the method for dilution effect.

Compared with sanger dideoxy chain-termination PCR sequencing PCR, pyrosequencing has quick, accurate, economic, inspection in real time The characteristics of survey；It does not need gel electrophoresis, does not need the label and the dyeing that carry out any special shape to DNA sample yet, has Very high repeatability；It is able to achieve the automation of the concurrency and height of height.

Two, the application progress of pyrosequencing techniques

1 application in single nucleotide polymorphism research

Single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNPs) is occurred in recent years Three generations's genetic marker, it refers to that there are two different bases on specific nucleotide position in genome, wherein least one kind It is not less than 1% in the frequency of group.SNPs is genetic polymorphism type most commonly seen in the genome of biology, it can be any one A series of labels are provided close to or within a gene to be studied；And the polymorphism in exactly this genome, i.e. genome sequence The difference of column constitute Different Individual and group to the neurological susceptibility of disease, to the science of heredity base of drug and environmental factor differential responses Plinth.

The research of SNP is main include two aspect: one is to the building of snp database, mainly discovery particular types biology All or part of SNP of genome.Second is that SNP functional study, discovery SNP is the first step of SNP research, and SNP function is ground Study carefully the purpose for being only SNP research.Sanger sequencing technologies have become the mainstream technology of extensive, accurate, quick discovery SNP.And Sequence verification analysis and frequency analysis are carried out to having SNP in database, is good at the pyrosequencing of short sequence and verifying Technology is to select well, carries out SNP research using pyrosequencing techniques, can more save the time and reduce consumption.

Nordfors etc. be respectively adopted Taqman fluorescent quantitation and pyrosequencing techniques to up to 1022 samples into The research of row SNP Genotyping, obtain it is identical as a result, this control experiment show pyrosequencing techniques be carry out it is high-throughput, Efficient, high accuracy the method for the SNPs of large sample.Wasson etc. carries out the pond DNA (DNA using pyrosequencing techniques Pools SNP gene frequency analysis).Rickert etc. carries out gene to 4 times of body potatos using pyrosequencing techniques Type research has 76 allele sites that can be identified with pyrosequencing techniques, effectively in 94 polymorphic site detections Rate is up to 81%.Jiang Siwen etc. identify using pyrosequencing techniques the work of porcine mtdna cytochrome b gene haplotype Make.Yuan Jianlin etc. carries out HLA-DRB genotyping research using pyrosequencing techniques, and experiment shows pyrosequencing As a result with Genotyping can be accurately carried out after the Gene sequence comparison of HLA database, this method can be applied to clinical organ transplant Donor/acceptor screening.

2 application in pathogenic microorganism Rapid identification

Jonasson etc. detects pathogen 16S rRNA gene 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 sequence, 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 A large amount of sample sequencing, concurrency and high efficiency highly significant are completed in short time.Gharizadeh etc. is with this technology to 67 A human papilloma virus 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 mutation research.Cheng Shaohui etc. extracts viral RNA from the Vero-6 cell of infection people SARS virus, using coke Multiple base mutation sites are sequenced phosphoric acid sequencing technologies and frequency of mutation analysis.Mutation is likely to occur by the way that sequencing analysis is 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 pleiomorphism of a different population is analyzed, and the CYPl7 gene pleiomorphism and prostate of African American are studied Relationship and clinical manifestation between cancer.Result of study shows that sequence is that the African American of the CYPl7 genotype of CC compares sequence Want high for the probability that the CYPl7 genotype African American of TT suffers from prostate cancer, it was demonstrated that the base in this kind of crowd is C's CYPl7 gene pleiomorphism and the disease incidence of prostate cancer are in close relations, are people at highest risk.Numerous clues show to be located at chromosome There are important relations for the COMT gene of 22q11 and schizoid morbidity, and the research work of scientist could not propose always Strong evidence；Shifman etc. proposes a kind of effective method, they utilize pyrosequencing techniques, to the moral of large sample It is that Jewish crowd carries out relevant single nucleotide polymorphism analysis, it was demonstrated that exist between schizoid generation and CMOT gene 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 PCR to mitochondrial DNA (mtDNA) analysis of variance, can not achieve to by containing pollutant, multiple The precise quantification of the mtDNA mixture of the compositions such as individual DNA is analyzed, and Andreasson etc. proposes one kind and mixes for mtDNA The novel quantization method based on pyrosequencing techniques of analysis is closed, it can be easily fast from court's exhibit mixing sample Speed accurately detects main and secondary mtDNA ingredient.Balitzki-Korte is using pyrosequencing techniques to line grain Body 12SrRNA gene carries 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 will be able to it is 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.

Three, pyrophosphoric acid sample adding device and sequencing device and system and development

The application of pyrosequencing techniques is dependent 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, primary structure all should include two parts: reactor part With faint light detecting portion.Reactor provide reaction carry out place, faint light detecting portion be responsible for detection reaction issue can It is light-exposed.In research and application process of the people to pyrosequencing techniques, the reactor of design and use can be mainly divided into 3 Class: micro plate reactor, micro-fluidic chip reactor and micro-array chip reactor.

And commercialized pyrosequencing instrument has emerged in foreign countries, however the report that domestic pertinent instruments itself are studied is very Few, there are no the appearances of corresponding product.One Typical Representative of external product is the PSQ96 of Pyrosequencing AB company, it It is the said firm's product released in 2001, system can carry out the independent sequencing of 96 tunnels or 384 road DNA samples simultaneously, work as survey Time generally used, accuracy and reliability reached 99%, has high pass at 45 minutes 1 hour when the of length no more than 300bp of sequence Amount, advantage quickly, economic.PSQ96 system has been widely used in basic medical research and clinical molecular diagnosis.

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

The problem of domestic instrument research at present is at the early-stage, and the road of production domesticization is very long, faces every aspect, is weighing After various hardware conditions required for sequencing system, finds the problem of primarily facing and challenge is the development of micro sample-adding subsystem Problem.

Four, in pyrosequencing sample adding system importance

Pyrosequencing techniques and products thereof are big flux, low cost, in due course, quickly and intuitively progress mononucleotide is more State property research and clinical examination provide ideal technical operation platform, are the genome times afterwards comprehensively to carry out gene sequencing The powerful of research.Pyrosequencing techniques are just received and are used by more and more researchers, 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 that pyrosequencing instrument is commercialized, the pyrosequencing techniques application Fang Xing in China It does not end.But at this stage, there are several restraining factors for the application and popularization of domestic pyrosequencing techniques: (1) existing quotient Product pyrosequencing instrument such as PSQ96, GS20 is expensive；(2) commercialized pyrosequencing service latency it is long and It is very inconvenient；(3) although some laboratories are in the research for carrying out the devices such as pyrosequencing chip, some laboratories at present There is homemade, the simple pyrosequencing experimental rig of structure, but domestic not towards low side, cheap, commercialization The Sequence Detection Instrument based on pyrosequencing techniques, be restrict pyrosequencing techniques application development critical 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 object, 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 larger rapidly, therefore template concentrations is caused to decline It is too fast.Due to the non-linear increase of response delay that diffusion generates, obtained fluorescent signals extend on a timeline, by force Degree reduces on the vertical scale, and eventually lead to serious curtailment nucleic acid is sequenced length.It is generally acknowledged that as caused by subsequent 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 the single sampling amount of permission can only be no more than in the reaction system of 50 μ L 0.3μL。

In addition, in addition to being loaded precision, sample-adding interval time accuracy is similarly important.Add only in constant duration DNTP needed for entering single cycle can just make the palliating degradation degree of each residual dNTP suitable, then the shadow caused by lower secondary response Sound also will be equal.The equal periods could provide benchmark to the signal in each period, be calculated convenient for subsequent according to fluorescence signal intensity The automated analysis of nucleotide combination number.

It is enriched although the loading device of production domesticization has compared, domestic existing micro sample-adding device has deficiency.Than If the sample-adding platform of day is answered in Shanghai, as large automatic sample pool processing equipment, it is able to carry out sample-adding, the vibration of 96 orifice plate of standard It swings, cleaning, but this set system is loaded micro- precision minimum and there was only 1 μ L, be unable to satisfy due to the limitation of nozzle processing technology The nL rank that pyrosequencing requires.By investigation, be limited to domestic application and manufacture level, the sample adding device of production domesticization all without Method meets the high request in pyrosequencing to sample-adding amount and repeatable accuracy.

Emblem Southeast China University Ge Jian etc. discloses one kind with faint light detection module and micro dNTP sample-adding module as crucial mould The liquid phase pyrophosphoric acid sample adding device and sequencing device and system of block, it is disclosed that the micro dNTP of a pressure control is loaded module, It can be achieved to be loaded while 96 road dNTP solution, minimum sample-adding amount is 1.2 μ L, worst error 13%；But signal noise is larger, It still needs to a more step and improves (Ge Jianhui etc., the development of the gene assaying device based on 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 using piezoelectric ceramics spray head, 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 stepper motor driving, sample-adding repeats essence Degree is greater than 95%, and single sampling minimum can reach 0.l μ L.It is micro- used in the above-mentioned preferable pyrophosphoric acid nucleic acid sequencing instrument of two classes It is more complicated to measure sample adding system structure, and sample needle is easily stifled, dNTP by different modes spray into sequencing reaction liquid not with Reaction solution contacts so that react with reaction solution undercompounding not exclusively, also higher to dNTP required amount and data are easily inaccurate； In addition, dismounting trouble, at high cost and be unfavorable for applying under specific condition.

Pyrosequencing (preosequencing) technology is the new DNA sequence analysis skill of one kind developed in recent years Art, the pyrophosphoric acid that it is discharged after being combined by nucleotide and template cause enzyme cascade, promote fluorescein luminescence and examined It surveys.It is an ideal genetic analysis technology platform, can not only carry out DNA sequence analysis, but also the list analyzed based on sequence can be carried out Nucleotide polymorphisms (SNP) detection and gene frequency measurement etc., 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) Cascade chemiluminescence reaction, reaction substrate 5 '-phosphosulfate (adenosine 5 ' phosphosulfate, APS) and fluorescence Element.Reaction system further 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 and template are matched, and polymerase can be incorporated into primer strand and release 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 issues the visible light signal directly proportional to ATP amount, and by Pyrogram^TMBe converted into a peak value, height with react The nucleotide number of incorporation is directly proportional.According to be added dNTP type and fluorescence signal intensity can logging template DNA in real time core Nucleotide sequence.Replace atriphos (dATP) with effectively with α-vulcanization atriphos (dATP α S) during the experiment It is utilized by archaeal dna polymerase, without being identified by luciferin.Since SpdATP α S can reduce the concentration of dATP α S catabolite, In recent years, single-stranded DNA binding protein (single starnd DNA binding portein, SSBP) and purifying Spisomer DATPaS's inhibits this active problem of bisphosphatase preferably to be solved using dATP α S catabolite, so that sequencing length Up to 10bp, the technology has been expanded in the application range of genetic arts.

In pyrosequencing, by using interim complementary strand synthesis reaction and chemiluminescence reaction, detection shines, thus To determine DNA sequence dna.By the reaction solution containing the pyrophosphoric acid and remaining nucleic acid primer being synthetically produced by complementary strand to carry 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 mobile It is fixed with the region for decomposing the enzyme of remaining nucleic acid primer, after remaining nucleic acid primer is decomposed, pyrophosphoric acid is made to be converted into ATP, Import chemiluminescence reaction slot.But the drawbacks of prior art is to must be added to a large amount of substrate and enzyme is reacted, anti-to guarantee It can should carry out completely, need to clean after reacting away extra substrate again later, this not only adds reaction process, increase every The reagents such as substrate can also be caused very serious waste, the reaction time is long, waste of manpower object by the cleaning and reaction difficulty of one step Power virtually reduces pyrosequencing popularization in the market and uses potentiality.

It is mostly at present that part of the manufacturer monopolizes manufacture and sale applied to the instrument of pyrosequencing, instrument is to match with reagent Set sale, cost is sufficiently expensive when sequencing, and Measuring error process is both needed to dependent on specific technical staff, and period length is at high cost, Volume needed for reaction is larger, more increases the cost of reaction, testing result is unstable, and repeatable accuracy is low.Therefore it designs autonomous Research and development develop a DNA sequencer suitable for pyrosequencing be very it is necessary to.

Five, the single-stranded isolation technics overview of DNA

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

The single-stranded separation method of common DNA is simultaneously existing insufficient as follows in biomedicine field:

1. thermal denaturation or alkali process.Double stranded PCR products are mainly carried out heating or alkali process by this kind of method, due to DNA Double-strand hydrogen bond under high temperature or a degree of alkaline environment can be broken, so that DNA becomes single-stranded.Although this kind of Method And Principle can Row, it is easy to operate, but this kind of method due to its separation rate and purity it is lower and gradually not as the purifying of single stranded DNA, be chiefly used in The double-strand of DNA separates.

2.T7 reverse transcription method.This kind of method is at an end of PCR primer 5 ' plus T7 promoter, to purify PCR amplification production Object 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 rate of DNA is higher, and the single-stranded purity of DNA obtained It is higher, but entire separation process need to be divided into two big steps and complete, and inconvenient, the time is longer, and needs strict control RNA enzyme Pollution, therefore there is certain limitation.

3. Exonucleolytic enzyme process (Higuchi and Ochman, Nucleic.Acids Res., 1989,17:5865).By It is phosphorylated in a PCR primer, PCR product when with exonuclease digestion, do not cut by the primer amplification chain being phosphorylated It cuts, enzyme is heated inactivation after digestion.This kind of method also needs purified pcr product, and separation program is tediously long, and operation is also inconvenient, and And the single-stranded pick-up rate of DNA depends 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 kind method 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 in column retention time 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 because have mispairing district there are due to more mutability, 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.Since a PCR primer is by biotin labeling, PCR amplification Chain in DHPLC, can and another common chain separate (Dickman and Hornby, Anal.Biochem., 2000,284: 164-167).This method can directly obtain that required DNA is single-stranded in 15min from double stranded PCR products, but this kind of method Implement to need mating sufficiently expensive instrument, therefore is difficult to popularize always.

5. magnetic capture method.Improvement and surface modification are carried out with surface of the nanotechnology to superparamagnetic nano particle Afterwards, it is prepared into superparamagnetism silica nanometer magnetic bead.The magnetic bead can specifically identify on micro interface with nucleic acid molecules and Efficiently combine.Using the superparamagnetism of silica nanosphere, in Chaotropic salt (guanidine hydrochloride, guanidinium isothiocyanate etc.) and outside Under the action of adding magnetic field, can from blood, animal tissue, food, pathogenic microorganism equal samples DNA and RNA separate, so It handles to obtain target with NaOH afterwards single-stranded.This kind of method is easy to operate, the used time is short, and entire process of extracting is divided into four steps, mostly may be used To complete in 36-40 minutes, and safe and non-toxic, without using toxic reagents such as benzene, chloroforms in conventional method, to experimental implementation The injury of personnel is reduced, and meets modern environmental protection concept, the magnetic bead specific binding single-stranded with DNA is so that the DNA extracted is single-stranded pure Degree is high, concentration is big, but coating magnetic bead used in this kind of method is costly, and needs to separate by magnetic frame, not only separates Higher cost, also inconvenience, therefore the popularization of the technology is limited to a certain extent.

6. asymmetric PCR.Above method is both needed to carry out additional processing after PCR, and asymmetric PCR can be in PCR amplification While preparation DNA it is single-stranded.Conventional asymmetric PCR uses the primer of two inequalities, is normally expanded in the circulation of beginning Increase.With the increase of circulation, measure few primer and gradually exhausted, and the primer of excess can continue straight line amplification generate DNA it is single-stranded (Gyllensten and Erlich,Proc.Natl.Acad.Sci.U.S.A.,1988,85:7652-7656).This kind of method Hybridization sensitivity and specificity with higher, and ease-to-operate is stronger, but the ratio of its primer needs to optimize, and non-spy The chance of different amplification increases, in addition, the single-stranded separation process of DNA is needed by electrophoresis, separation program is complicated, and electrophoresis is often seen Smear, it is time-consuming and be not obvious.

Above-mentioned several separation methods all have certain limitation, therefore, in order to meet operating to the single-stranded separation of DNA Property and cost-effectiveness requirement, DNA in the prior art it is single-stranded using integrated extraction work station, will be with streptavidin In conjunction with DNA double chain, there is work station affine connector suction filtration needle and matched pump, the affine connector of DNA in conjunction with after to pass through Suction filtration, which is adsorbed on, filters filter membrane lower part in needle, and work station is furnished with track and related system, will filter needle after suction filtration and moves to Sheng Have in the disk of NaOH, by alkali process solution double helix, it is single-stranded to obtain DNA, cleans and collects after filtering again.Filter general 24, needle (4*6) is one group, when use must assure that sample or the reagent of sufficient amount to guarantee the normal operation of work station, therefore it is this DNA single-stranded collection mode is very not flexible, work station can only be added with fixed amount and work, and a large amount of loss is more It being generated in secondary suction filtration and transfer process, this is very unfavorable to micro-collection, and filters needle group and need while working, this All there is certain volume requirement to each component of work station, entire work station occupied space is very big.Huge system makes in DNA In single-stranded separation operation process, micro- splitter needs liquid relief back and forth, and operation is very complicated, and not only separation cycle is long, efficiency It is low, and integral device price is more expensive, leads to the costly of the single-stranded separation of DNA, it is also necessary to a large amount of reagent and other resources are expended, It is extremely uneconomical.In addition, it is metal material that needle is filtered in the work station, and it is expensive, it is re-used after often handling, therefore easily Lead to the cross contamination between residue, reliability is not high, will cause certain do to the accuracy of separation and testing result It disturbs and influences.And it is adherent during solution extraction to have portion of residual solution, it cannot be micro- so that a certain amount of target DNA is single-stranded Splitter absorption, causes the DNA proportion of single chain obtained to reduce, affects separation rate, cause waste.Therefore, it is used for pyrophosphoric acid The single-stranded separation problem of the DNA of the high quality and high efficiency of sequencing is urgently to be resolved.

Summary of the invention

In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide it is a kind of it is easy to operate, quickly detect Pyrosequencing sample adding device and system.

For achieving the above object, The technical solution adopted by the 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 packet At least one Disengagement zone DNA is included, the Disengagement zone includes the internal hollow Filter column for being equipped with filter membrane, is connected with affine connector DNA is single-stranded and is not connected with the DNA of affine connector and single-stranded separates after the membrane filtration；

The reaction zone includes sample-adding portion and sample cell, and the sample-adding portion includes dNTP reagent trough, sample-adding punch block and installation Multiple sample needles thereon, the separator disk are located at the upper side of reagent trough；The sample-adding punch block is located on sample cell；It is described Sample cell is equipped with multiple slot positions.

As a further improvement of the above technical scheme:

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

Further, as a preferred embodiment, the outer diameter of Filter column is equal to the internal diameter of collecting pipe, pass through frictional force Filter column and collecting pipe can be made to be maintained at a relatively steady state, not needing plus structural is that Filter column limit admittedly It is fixed.

Another preferred mode, the separator disk are equipped 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 equipped with dismountable upper cover, and the upper cover is connect by connecting band with collecting pipe.

Single stranded DNA after separation is connected with the single-stranded of affinity body on filter membrane, needs this chain that can wash to filter membrane It is de-, containing another complementary strand without affine connector in filtrate, when needing backward sequencing can to the single stranded DNA in filtrate into Row is collected.When needing to collect with affine connector the chain on filter membrane, the collecting pipe of recycling is can be used in collecting pipe, Collecting pipe only plays the role of Recycling of waste liquid at this time, and recycling use reduces cost, and environmental protection reduces the generation of white pollution.

Preferably, the reagent trough is equipped with multiple reaction positions, and the side-lower of the reagent trough is equipped 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 moving portion Between move reciprocatingly；The sample-adding punch block is made rotating motion by shaft.

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

Preferably, the shaft is equipped with driving 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 further includes dry slot and rinse bath.

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

It is a further object of the present invention to provide a kind of pyrosequencing devices, including any of the above-described sample adding device with unification Detection zone forms, and the detection zone includes bioluminescent detection device, and the slot position is connect with 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 slot positions and encloses on the central axes set.Camera can shoot the reaction in all slot positions by rotation Situation.

The object of the invention is also to provide another pyrosequencing systems, 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 includes to solution concentration and pH value Control.

In view of the above-mentioned problems existing in the prior art, the advantage of pyrosequencing of the present invention sample adding device is:

(1) it is extracted by way of filter centrifugation by DNA is single-stranded, the economization for realizing the collection of a sample one is small Typeization is collected, and the single-stranded separation method of rapid DNA and device of a kind of efficiently low loss are provided.This method is simple to operation, obtains It takes the target sample time short high-efficient, can be used for trace amount DNA single-stranded collection and extraction, sample loss can almost be ignored, make With reagent is few, requirement to equipment is low, it is not necessarily to suction pump in separation process, device configuration is enormously simplified, it is total to reduce equipment Cost is effectively simplified operating procedure, shortens the operating time, reduces working strength, improves work efficiency；Using with it is normal The matched Filter column of rule centrifuge tube not only ensure that the quality and efficiency of separation, but also separation process is simply easily realized, filtering The filter membrane of column is separated by the way that physics mode is single-stranded to DNA, reduces the difficulty and 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 type of the single-stranded separator of DNA, under the premise of ensuring identical function, increases The application occasion for having added the single-stranded separator of DNA of the invention, avoids that product structure is single, and adaptability significantly increases；And Structure symmetrical above and below can be used, can exchange and be used cooperatively up and down.The single-stranded separator of this DNA is moulded using cheap PC Material, economic and practical, the design of infundibulate inner cavity are conducive to the aggregation and guidance of reaction solution, so that reaction solution separation is more abundant More completely, the target DNA for obtaining higher proportion is single-stranded, ensure that higher separation rate, avoids 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 versatility of this DNA is extremely strong, is applicable in Face is extremely wide, therefore its application prospect is very wide.

(2) the micro to sample of dNTP reagent can be realized using the simple sample needle of a structure in the present invention, has abandoned transmission Hollow needle tube take out spray formula give sample loading mode, only by the adsorption capacity between the sample needle and liquid of the sample needle and its in difference The control of movement speed difference in liquid can be realized microsampling sample-adding, and only by sample needle in sequencing reaction liquid on Stirring can be realized for several times for lower movement so that the more complete result of enzyme reaction is accurate, this loading methods and its device are suitable for any In detection device, disassembly is easy, and cleaning is simple and convenient, and a variety of sample adding devices in the prior art such as stifled needle will not occur not Foot, sample-adding repeatable accuracy are greater than 95%, and minimum sample-adding amount is up to 0.1 μ L, and it is good to be loaded homogeneity, the sequencing time shorten dramatically and As a result accuracy is high；Furthermore qualitative and quantitative detection can be carried out to sample nucleic acid sequence by detection device associated with institute；Therefore Its application prospect is very wide.

(3) pyrosequencing sample adding device of the invention manipulates whole process, no infection, accuracy rate using manipulator Height has improved efficiency and precision.

In short, pyrosequencing sample adding device provided by the invention and system reduce the dosage of substrate and enzyme system, inspection It is accurate to survey, and reaction 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 quotient, price is greatly reduced, and sample adding device and sequencing device and system structure are accurate, It is low to the dosage requirement of DNA fragmentation to be measured and substrate, the testing cost of pyrosequencing is reduced, pyrosequencing is expanded Use scope.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of pyrosequencing sample adding device of the invention.

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

Fig. 3 be provided by the present invention for pyrophosphoric acid the Disengagement zone DNA one using preferred embodiment.

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

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

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

Fig. 7 is the structural schematic diagram 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 the sequence spectrum measured in embodiment using the sample adding device provided by the present invention for pyrosequencing system Figure.

Figure label explanation:

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

Specific embodiment

The present invention is made further to illustrate in detail, completely below 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 and test and analyze DNA sequence dna, and DNA sequence dna to be sequenced is target sequence, by target DNA sequence dna 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 mounted in analysis station 6, sample area, reaction zone and inspection It surveys area and passes through control zone monitoring, control.Entire analytic process uses manipulator operation.

It before carrying out pyrosequencing, needs first to expand the DNA profiling with sequencing, to reach amplification requirement DNA concentration.When amplimer designs, affine connector is had on amplimer, affine connector is preferably biotin, affine company Junctor preferably marks on one end primer of target DNA, and PCR amplification is carried out using the 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, and separator disk 2 includes at least one Disengagement zone DNA, and the Disengagement zone DNA uses physical filtering Mode, Disengagement zone includes the internal hollow Filter column 211 for being equipped with filter membrane 22, and filter membrane 22 is high molecular nano-microsphere structure, It is since the aperture between high molecular nano-microsphere is less than the diameter of affine connector, the DNA with affine connector label is single-stranded Stay on film, according to sequencing need to collect tape label DNA is single-stranded or its complementary strand, be used for pyrosequencing.

In the present embodiment, separator disk 2 is equipped with multiple collecting pipes 23, and Filter column 21 is installed in collecting pipe 23, and filter membrane 22 In the end of one end of Filter column 21.The outer diameter of 21 lower section of Filter column is identical as the internal diameter of collecting pipe 23.

Double-stranded DNA will stay on filter membrane 22 after alkaline hydrolysis to affine connector chain, and collecting this chain only need to be in mistake It is collected after collection liquid is added in filter column 21；If you need to collect complementary strand, the complementary strand in filtrate is received after filtrate being collected Collection.

As shown in figure 3, collecting pipe 23 is used to collect the waste liquid that generates when being centrifuged, need to topple in time after each step to prevent Only cross contamination, the upper pipes of collecting pipe 23 are cone as cylindrical body, lower end, and bottom is dome shape.As another excellent The embodiment of choosing, collecting pipe 23 configure upper cover 231, and upper cover 231 is detachably connected to collecting pipe 23 by connecting band 232 On, due to the connection of connecting band 232, upper cover 231 can also closely be fastened on Filter column 21 or collecting pipe 23 after being packed into Filter column 21 On, the effect of upper cover 231 is to prevent from liquid splash from going out Filter column 21 in liquid centrifugation to cause damages and pollute.

Preferred 22 material of filter membrane of institute is polyethylene microballoon in the present embodiment, and hole is preferably 10 μm between microballoon, is less than The diameter of affine connector will directly 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, 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 does not move in piping and druming or centrifugal process, the present embodiment In further preferably above filter membrane 22 be equipped with film pressing device, film pressing device includes gasket and/or press mold frame.The liquid of purifying to be separated Body contacts after first passing through gasket with filter membrane 22, and gasket is preferably fibrous material, can tolerate soda acid and most of organic solvent, to big Partial biomolecule will not generate absorption.

Preferably in the top of gasket, 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, move filter membrane 22 will not in the use processes such as piping and druming or centrifugation It is dynamic, it causes to collect and lose.

Reaction zone includes sample-adding portion and sample cell 3, and sample-adding portion successively includes dNTP reagent trough 7, dry slot 71, rinse bath 72, 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 at the upper side of reagent trough 7.Add Sample punch block 4 is disc, and sample-adding punch block 4 can be moved reciprocatingly by moving portion between reagent trough 7 and sample cell 3.Moving portion Including the sliding rail 62 for being fixed on 6 top of analysis station by mounting rack 61 and the sliding block for being loaded 4 bottom end of punch block.Dry slot 71 is true Empty dry section.

Reagent trough 7 is set there are four position 74 is reacted, and the side-lower of reagent trough 7 is equipped with substrate supply unit 73, substrate supply unit 73 The different nucleic acid primers of dNTP are conveyed to reaction position 74 by conveyance conduit 1.Reaction position 74 to be measured is for holding DNA to be sequenced Sequence, substrate supply unit provide dNTP and provide environmental basis with target DNA hybridization reaction for hybridization reaction.For pyrosequencing DNTP include tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP, substrate with target DNA for hybridize, and needs are reacting Related enzyme systems catalysis reaction is added in system to carry out, specially archaeal dna polymerase, carries out complementary strand synthesis reaction, closed in complementary strand At when obtained by-product pyrophosphoric acid be converted to ATP, react ATP and luciferin in the presence of luciferase, carry out luminous.Such as Complementary strand synthesis occurs for fruit, then generates pyrophosphoric acid, as a result shine, therefore by monitoring to it, can learn that generation is complementary The base type that chain synthesis, i.e. group enter, thereby determines that DNA sequence dna.

Sample-adding punch block 4 is made rotating motion by shaft 43, and shaft 43 is equipped with driving motor.Sample needle 411 is solid needle, is used In transfer dNTP reagent, sample needle 411 is fixed in the through-hole of sample-adding punch block 4.By the single-stranded requirement according to reaction system of DNA It is transferred in reaction position 74, sequentially adds dNTP after enzyme system is added, addition sequence is unlimited, and for each site, four kinds of substrates are each It is added once, substrate is provided by substrate supply unit 73.

Detection zone includes bioluminescent detection device 5, and slot position 31 is connect with bioluminescent detection device 5.As closed after reaction It at complementary strand, then shines, bioluminescent detection device 5 judges whether the site is reacted and shone for detecting, and then sentences The base sequence in the disconnected site out.

Sample-adding punch block 4 is located on sample cell 3；Sample cell 3 is equipped with multiple slot positions 31, has sequencing reaction liquid in slot position 31. Slot position 31 is evenly distributed in a series of concentric circles along the center of circle of sample cell 3, and slot position 31 is made of transparent material, and slot position 31 extends 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 central axes that all slot positions 31 are gathered.Camera is commercially available iKon-M deep refrigerating 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；Using the sample of above-mentioned sample adding device detection known array (sequence CAATATTCGCCAGGT), wherein 41 diameter of sample needle is 1.5mm, and surface smoothness is Ra 0.8；Loading methods packet Include: step a makes 41 periphery of sample needle adhere to the dNTP reagent by the dNTP reagent that sample needle 41 is inserted into sample cell 3；Step The sample needle 41 for adhering to the dNTP reagent is inserted into slot position 31, then sample needle 41 is made to leave sequencing reaction liquid by rapid b；It is described Movement speed when sample needle 41 described in step a leaves the dNTP reagent solution is 10cm/s, is loaded described in the step b Movement speed when needle 41 leaves the sequencing reaction liquid is 1cm/s, leaves sequencing after moving up and down 8 times in sequencing reaction liquid Reaction solution.

Fig. 9 uses the resulting sequencing spectrogram of pyrophosphoric acid nucleic acid sequencing detection device for the present embodiment, as shown in Figure 9, wherein The sequence TCATATTCGCCAGT of dNTP is sequentially added, wherein non-appearance and subsequent not being inconsistent with actual sequence when T being added for the first time DNTP reagent solution also non-appearance (being marked in Fig. 9 with circle, i.e. T, T, C), the sequence measured is CAATATTCGCCAGGT, Completely the same with sample actual sequence, accuracy 100% detected the time of single base less than 1.5 minutes, this sequence is about It is 25 minutes time-consuming, and minimum sample-adding amount is up to 0.1 μ L.

The set-up mode of pyrosequencing of the invention sample adding device can preferably transmit substrate and DNA, reduce and pass Loss during passing.

The shape of reaction zone is unlimited, and the specific structure in sample adding device and sequencing device and system can be according to the need of detection It is designed adjustment, any planform for being able to achieve pyrosequencing and relative position are deemed to fall of the invention Within protection scope.

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

Enzymatic mixture includes:

Substrate mixture includes:

APS 0.4mg/L；

Firefly luciferin 0.4mmol/L.

In reaction process, reacts, needed after reaction pair in each step selection optimum pH value for enzymatic activity PH value in reaction system is adjusted the reaction condition ordinary skill to adapt to other reactions, in specific reaction system Personnel can obtain according to the prior art.For example, when including apyrase in washing step to remove excessive nucleosides Sour type and when ATP, because of the continuity of processing step, buffer can be used together with apyrase, pH The clean property level of apyrase can be made to optimize.Then, polymerase is used in next nucleotide incorporation step, The different buffers with the best pH condition for polymerase can be used, to optimize the clean property 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 CAATATTCGCCAGGT, wherein sequentially adding the sequence of dNTP For TCATATTCGCCAGT, wherein non-appearance and the subsequent dNTP reagent not being inconsistent with actual sequence also do not go out when T being added for the first time Peak, the sequence measured are CAATATTCGCCAGGT, accuracy 100% completely the same with sample actual sequence；Detection is single The time of a base, this sequence was about 25 minutes time-consuming less than 1.5 minutes, and minimum sample-adding amount is up to 0.1 μ L.

Sequencing result is judged that other devices that can detecte bioluminescence can be used as inspection by bioluminescence Device is surveyed, herein with no restrictions.

The pyrosequencing sample adding device based on pyrosequencing in embodiment of the present invention, further includes each sample Liquid supply and passing away between storage device, liquid supply passage is for conveying reagent and sample to be reacted, liquid Passing away is used to the liquid after reaction or after centrifugation being expelled to collecting part.

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 sample adding device and sequencing device of the invention and system in the reaction, need to guarantee each portion's operation in optimum In the environment of carry out, enzyme system needs to be reacted in the highest reaction system of activity, to guarantee fully reacting, therefore control zone In be equipped with can guarantee these reaction be capable of effective carry out several components, including centrifuge, pH meter, heating component, control The standing structure of the sequencing equipments such as signal transmitting assembly processed.

The system and method for embodiment of the present invention may include carrying out some designs using computer-readable medium, dividing Analysis or other operations, such media storage have the instruction for executing on the computer systems.For example, the detected signal of processing And/or analysis some embodiments of the data of sequencing result system and method generation, wherein processing and analysis embodiment It executes on the computer systems.

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

Display may include providing the display of visual information, and such information is usually logically and/or physically organized As pixel array.It may also comprise interfacial level controller, wherein may include that any different oneself knows or following software program, be used for Offer outputs and inputs interface." graphic user interface (the Graphical User for example, interface may include so-called Interfaces, commonly referred to as GU I) ", one or more images can be provided for user and shown.Use the common skill of related fields Art personnel are known to select or input mode, and interface usually can receive user's input.

In identical or alternate embodiment, it includes that so-called " Command Line Interface " is (logical that application on computers, which can be used, Frequently referred to CLI) including interface.CLI is usually provided based on the text applied with interacted between user.In general, order line circle Face is shown in the form of line of text by display to be exported and receives input." the order for example, certain implementation procedures may include so-called Row interpretive program (shell) " the known Unix command line interpreter (Unix of such as person of ordinary skill in the relevant Shell), or using object oriented program architecture Microsoft Windows Powershell, such as Microsoft.NET frame.

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

Processor usually executes operating system, and any operating system in the prior art may be selected in operating system, as long as this Field technical staff can be used for the work such as processing detection result and data and think to can be included within protection scope.

The pyrosequencing sample adding device based on pyrosequencing in the present invention can be widely used for DNA sequence dna Determine, diagnosis, check, determination, diagnosis of SNP site etc., it can be achieved that certain sample while be sequenced, condition of the principle to sequencing It is required that it is low, the experiment reagent for additionally increasing the valuableness such as excitation light source and fluorescer is not needed, it can be achieved that easy cheap DNA is surveyed Sequence work, and testing result is stablized, accuracy rate is high, and overcoming previous equipment cannot sufficiently carry out or complementary strand synthesis reaction The drawbacks of first excessively carrying out, reaction volume is small, can complete the work of the stage of reaction within the shorter time.

Using pyrosequencing sample adding device provided by the invention and the analysis method of system, include the following steps, In undisclosed partially can refer to the prior art:

(1) combine: by the DNA fragmentation after amplification in conjunction with sepharose 4B, the length of DNA fragmentation is 10~20kb, DNA minimum applied sample amount should be not less than 500ng, and sepharose 4B diameter is 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: drawing the DNA double chain after combining into Filter column 21, Filter column 21 is previously charged into collecting pipe 23, is put Enter and 1min is centrifuged with 12000rmp after centrifuge, removes excess of solvent；Wherein collecting pipe 23 is 1.5mL centrifuge tube；Filter column 21 For upper end opening, the semi-enclosed cylindrical body in lower end, 21 upper end opening outer rim of Filter column is equipped with boss, to be fixed on collecting pipe 23 On, 21 lower end of Filter column is built-in with filter membrane 22, and 21 diameter of Filter column is 4.5mm, and 22 diameter of filter membrane is 4.7mm, by that will filter Film 22 is pressed into be allowed to fit closely in Filter column 21 by force and leave no gaps；

(3) it washs: appropriate 70~80% ethyl alcohol is being added, is washing away the amplifing reagents such as remaining Taq enzyme, gently piping and druming is mixed 12000rmp is centrifuged 1min after even；

(4) alkaline hydrolysis: being added 0.4M NaOH and 1M NaCl unlocks double-stranded helical, and 12000rmp is centrifuged after gently piping and druming mixes 1min；

(5) it adjusts pH value: appropriate elution buffer or ultrapure water cleaning is added, washes away remaining NaOH, equilibrium ph is into Property, 12000rmp is centrifuged 1min after gently piping and druming mixes；

(6) it collects: collection liquid such as ultrapure water etc. is added, is gently blown and beaten after addition to the single-stranded complete suspension of DNA, is used after suction It is sealed in pyrosequencing or 4 DEG C.

(7) it is loaded: any dNTP reagent being dipped by sample needle 41 and is made described in 41 periphery of the sample needle attachment DNTP reagent；

(8) it reacts: the sample needle 41 for adhering to the dNTP reagent being inserted into 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, preferably by 4 kinds of dNTP reagents it is any one or more depending on sequence to be measured with which kind of Sequence is added in sequencing reaction liquid, such as: when only detecting single base and whether making a variation, can first it be determined according to known context After base positions to be measured, it is repeated in above-mentioned steps 4 times sample-addings (4 kinds of dNTP reagents are added), is separately added into same sequencing reaction The base is detected in liquid.

Since the diameter of affine connector is greater than 22 diameter of filter membrane of the single-stranded separator of DNA, pass through DNA is single-stranded When filter membrane 22, be not associated with affine connector DNA is single-stranded and other impurities can combine affine connector by filter membrane 22 Single-stranded be left on film and can not pass through, this filtering is physical.

Solution involved in DNA separation process in the present embodiment, parameter and other separation conditions, are the present embodiment In preferred embodiment, it is any that referring to the prior art can to play experiment condition, parameter and the solution of respective action available Process is isolated and purified involved in the present invention, the design parameter and solution selection in the present embodiment should not be used as to the present invention Limitation.

Sequencing spectrogram obtained by the present embodiment and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 96%.

Embodiment 2

Since the chain collected on filter membrane 22 needs to be sucked out or poured out, such collection mode may cause one Fixed collection loss, therefore inventor preferably sets both ends for Filter column 21 and exchanges the bipitch structure used, filter membrane 22 is set In the non-end face of Filter column 21, when needing to collect, usual manner, such as centrifugation are used after 21 both ends of Filter column are exchanged, It can elute all DNA on filter membrane 22 is single-stranded, reduce sample loss.

As shown in figure 5, the present embodiment and the difference of embodiment 1 are only that, the Filter column 21 in embodiment 2 is symmetrical above and below The hollow form cylindrical body of structure, filter membrane 22 are vertically positioned in the middle part of hollow cylinder, the Filter column 21 can both ends exchange and use, hollow posts The internal diameter of body is 4.5mm, and 22 diameter of filter membrane is 4.7mm, is allowed to fit closely by the way that filter membrane 22 to be pressed into Filter column 21 by force It leaves no gaps, filter membrane 22 is added in cylinder and limits squeeze-film mechanism (not shown), can not be displaced with fixing filter membrane 22.By In the single-stranded separator of the DNA, still functionally both ends are all the same either in structure, thus the single-stranded separator of the DNA without Liquid feeding end and outlet end need to be distinguished, both ends can arbitrarily select a use when in use.

After washing away extra NaOH by step (4), the both ends of Filter column 21 are exchanged, collection liquid is added, it is static 1mins or more can carry out elution step without piping and druming, and centrifuge speed is no more than 10000rpm when elution, at least be centrifuged 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) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 95%.

Embodiment 3

As shown in fig. 6, the present embodiment and the difference of embodiment 1 are only that, the mistake of the single-stranded separator of DNA in embodiment 3 Filter column 21 uses space circular platform type structure, and 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 column 21 is consistent with the diameter of filter membrane 22, and 22 two sides of filter membrane are equipped with squeeze-film mechanism (not shown), with fixed filter Film 22 can not be displaced.The side wall of the open lower end of truncated cone-shaped Filter column is equipped with boss, guarantees to consolidate timing at both ends It is scheduled on the boss of 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 The difference for applying example 1 is only that: after washing away extra NaOH by step (4), being exchanged the both ends of Filter column 21, is added and receives Liquid collecting, static 1mins or more can carry out elution step without piping and druming, and centrifuge speed is no more than 10000rpm when elution, until It is centrifuged 2min less.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) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 95%.

Embodiment 4

As shown in fig. 7, the present embodiment and the difference of embodiment 1 are only that, in order to preferably assemble and guide reaction solution, make Reaction solution separation more sufficiently more completely, evades filtering dead angle, which 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 to 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 two sides of filter membrane are equipped with squeeze-film mechanism and (do not show in figure Out), it can not be displaced with fixing filter membrane 22, the middle part of two Filter columns 21 is equipped with boss, guarantees that both ends can consolidate 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 The basal diameter of structure, rounding end is consistent with 21 internal diameter of Filter column, and top surface diameter is consistent with the diameter of split tunnel 211, should The aggregation and guidance of kind structure being provided with conducive to reaction solution, so that reaction solution separation is more sufficiently more completely.Therefore, the embodiment In the single-stranded separator of DNA make on the basis of embodiment 2 DNA it is single-stranded be separated by filtration more thoroughly, separative efficiency significantly mentions It is high.

The step of using the separation method of separator affiliated in the present embodiment with described in embodiment 1, is identical, with reality The difference for applying example 1 is only that: after washing away extra NaOH by step (4), being exchanged the both ends of Filter column 21, is added and collects Liquid, static 1mins or more can carry out elution step without piping and druming, and centrifuge speed is no more than 10000rpm when elution, at least It is centrifuged 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) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 95%.

Embodiment 5

The present embodiment and the difference of embodiment 4 be only that, filter membrane 22 can be placed in one any bit perpendicular to split tunnel 211 It sets, two Filter columns 21 are hollow dissymmetrical structure, and the diameter of filter membrane 22 is slightly larger than split tunnel 211, and 22 two sides of filter membrane are set There is squeeze-film mechanism (not shown), can not be displaced with fixing filter membrane 22.

When design of primers, the affine connector label that biotin-avidin combines also may be selected, it is any to carry out DNA The connector of label is used equally for being fixed on film, and this will not be repeated here.

The affine connector of Avidin is had as used, then the single-stranded isolated membrane system of DNA also can choose with affine suction Attached membrane system, it is single-stranded for separating DNA.Membrane system with affine suction-operated includes silicon fiml matrix membrane, Magnetic Granular Films, yin Ion exchange material film, nitrocellulose filter or PA membrane, and through modification, coated magnetic bead and/or silicon dioxide film etc., Herein with no restrictions.

Regular mode in the prior art, such as the single-stranded separation of DNA can also be used in double-stranded DNA separation in the present invention Kit etc., as long as can play the single-stranded structure separated and collected of DNA and method should all be included within protection scope of the present invention, This will not be repeated here.

Sequencing spectrogram obtained by the present embodiment and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 95%.

Embodiment 6

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

Sequencing spectrogram obtained by the present embodiment and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 96%.

Embodiment 7

The present embodiment and the difference of embodiment 1 are only that: the end face of the sample needle 41 be cone, diameter 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 examination Movement speed when agent liquid is 5cm/s, and sample needle 41 described in the step b leaves the movement speed when sequencing reaction liquid For 4.5cm/s, sequencing reaction liquid is left after moving up and down 12 times in sequencing reaction liquid.

Sequencing spectrogram obtained by the present embodiment and other experimental result datas (sequencing time, sample-adding repeatable accuracy, homogeneity) With the consistency that 1 the data obtained of embodiment is within the scope of theoretical error, sample-adding repeatable accuracy is 95%.

Be it is necessary to described herein finally: above embodiments are served only for making technical solution of the present invention further detailed Ground explanation, should not be understood as limiting the scope of the invention, those skilled in the art's above content according to the present invention The some nonessential modifications and adaptations made all belong to the scope of protection of the present invention.It is it is necessary to described herein finally: with Upper embodiment is served only for being described in more detail technical solution of the present invention, should not be understood as to the scope of the present invention Limitation, some nonessential modifications and adaptations that those skilled in the art's above content according to the present invention is made belong to Protection scope of the present invention.

Claims (9)

1. pyrosequencing sample adding device, which is characterized in that including sample area and reaction 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 It (2) include at least one Disengagement zone DNA, the Disengagement zone includes the internal hollow Filter column (21) for being equipped with filter membrane (22), institute It states separator disk (2) and is equipped with multiple collecting pipes (23) for being used to collect the waste liquid generated when centrifugation, the Filter column (21) is installed on In collecting pipe (23), the filter membrane (22) is located at the end of Filter column (21) one end or the non-end of Filter column (21)；It is connected with parent With the DNA of connector it is single-stranded and be not connected with affine connector DNA it is single-stranded through the filter membrane (22) filtering after separate；

The reaction zone includes sample-adding portion and sample cell (3), the sample-adding portion include dNTP reagent trough (7), sample-adding punch block (4) and The multiple sample needles (41) being mounted thereon, the separator disk (2) are located at the upper side of reagent trough (7)；The sample-adding punch block (4) On sample cell (3)；The sample cell (3) is equipped with multiple slot positions (31).

2. pyrosequencing sample adding device according to claim 1, which is characterized in that the collecting pipe (23) is equipped with can The upper cover (231) of disassembly, the upper cover (231) are connect by connecting band (232) with collecting pipe (23).

3. pyrosequencing sample adding device according to claim 1, which is characterized in that the reagent trough (7) is equipped with more The side-lower of a reaction position (74), the reagent trough (7) is equipped with substrate supply unit (73), and the substrate supply unit (73) passes through defeated Send pipeline (1) to tetra- kinds of nucleic acid primers of dATP, dCTP, dGTP, dTTP of reaction position (74) conveying dNTP.

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

5. pyrosequencing sample adding device according to claim 4, which is characterized in that the moving portion includes passing through peace Shelve the sliding block of sliding rail (62) and described sample-adding punch block (4) bottom end that (61) are fixed on above analysis station (6).

6. pyrosequencing sample adding device according to claim 4, which is characterized in that the sample needle (41) is solid Needle, the sample needle (41) are fixed in the through-hole of sample-adding punch block (4).

7. pyrosequencing sample adding device according to claim 1, which is characterized in that slot position (31) is along sample cell (3) The center of circle in a series of concentric circles it is evenly distributed, the slot position (31) is made of transparent material, and the slot position (31) extends into life Object luminescence detection apparatus (5).

8. a kind of pyrosequencing device, it is characterised in that: including any sample adding device of claim 1~7 with unification Detection zone forms, and the detection zone includes bioluminescent detection device, and the slot position is connect with bioluminescent detection device.

9. a kind of pyrosequencing system, it is characterised in that: including sequencing device according to any one of claims 8 and one and for controlling The control zone of sequencing device processed.