CN107312710B - DNA sequencing device based on pyrosequencing and sequencing method thereof - Google Patents

Abstract

The invention provides a DNA sequencing device based on pyrosequencing, which comprises a sample area, a reaction area and a detection area; the sample area comprises a rotatable separation disc, and the separation disc comprises a filter column with a filter membrane arranged inside; the reaction area comprises a sample adding frame, dNTP reagent tanks and a reaction tank, a plurality of sample adding needles are fixedly arranged on the sample adding frame, the sample adding frame makes reciprocating motion between the dNTP reagent tanks and the reaction tank through a linear moving device, a rotating shaft is arranged at the center of the sample adding frame, the rotating shaft drives the sample adding frame to rotate through a motor, and the sample adding frame makes lifting motion along the rotating shaft through a lifting device; the reaction tank is provided with a plurality of reaction sites, and the reaction sites extend to the detection area; the detection zone includes bioluminescence detector and revolving stage, and bioluminescence detector detachable installs on the revolving stage, and the revolving stage passes through the motor and drives bioluminescence detector rotation. The DNA sequencing device based on the pyrosequencing has the advantages of simple and convenient operation and rapid detection.

Description

DNA sequencing device based on pyrosequencing and sequencing method thereof

Technical Field

The invention relates to the technical field of DNA sequencing, in particular to a DNA sequencing device based on pyrosequencing, and belongs to the field of gene detection.

Background

Mono, pyrosequencing Profile

Pyrosequencing (Pyrosequencing), which was developed in 1987 and is based on the detection of pyrophosphate (PPi) released during DNA synthesis, generates visible light in proportion to the number of deoxynucleoside triphosphates (dntps) polymerized under the catalytic action of a series of enzymes, and detects the DNA sequence by the detection of visible light. There are two methods of implementing pyrosequencing: liquid Phase Pyrosequencing (Liquid Phase Pyrosequencing) and Solid Phase Pyrosequencing (Solid Phase Pyrosequencing).

Liquid-phase pyrosequencing is an enzyme cascade chemiluminescence reaction in the same reaction system catalyzed by 4 enzymes, and the principle is as follows: after the primer is annealed with the template DNA, under the synergistic action of DNA polymerase (DNA polymerase), ATP sulfurylase (ATP s mu Lfurylase), luciferase (1uciferase) and Apyrase (Apyrase), the polymerization of each dNTP on the primer DNA is coupled with the release of a fluorescence signal, and the purpose of real-time determination of a DNA sequence is achieved by detecting the release and the intensity of fluorescence (Mayongping et al, a pyrosequencing technology and the application thereof in the field of molecular biology [ J ]. molecular biology, 25(2): 115-.

The reaction system for liquid-phase pyrophosphate sequencing consists of a reaction substrate, a single strand to be detected, a specific sequencing primer and an enzyme, wherein the reaction substrate is 5' -phosphoryl sulfate (APS) and fluorescein (1 uciferin). The liquid-phase pyrosequencing reaction process is a process of adding 4 kinds of dNTPs into a reaction system in turn to participate in reaction, and only one kind of dNTP participates in each reaction. If the added dNTP is just capable of pairing with the next base of the DNA template, it is added to the 3' end of the sequencing primer under the action of DNA polymerase, and releases one molecule of pyrophosphate (PPi); under the action of ATP sulfurylase, the generated PPi and APS are combined to form ATP; under the action of luciferase, the generated ATP is combined with luciferin to form oxyluciferin, and visible light is generated. If the dNTP is added to match with n consecutive identical bases on the bottom of the DNA template, the visible light intensity released is n times that of the case where only 1 base is matched, as known from the reaction equation, i.e., the light intensity released during the reaction is proportional to the number of matched bases. If the added dNTP does not match the next base of the DNA template, the reaction does not occur and no visible light is released. dNTPs and ATP that have not reacted are degraded by the action of the nucleotide degrading enzyme Apyrase.

The visible light released from each reaction is converted by a weak light detection device, then processed into a digital signal, and processed by PC software to obtain a specific detection peak, wherein the height of the peak is in proportional relation with the number of matched bases.

After the previous reaction, another dNTP is added, and the reaction is repeated. And finally, the readable DNA sequence information to be detected can be obtained according to the obtained light intensity peak value diagram.

It should be noted that: deoxyguanosine monophosphate (dAMP), a degradation product of dATP, is an inhibitor of luciferase, and the concentration of the dAMP is higher and higher as the reaction progresses, so that the continuation of the pyrosequencing chemiluminescence reaction is prevented. This is also the main reason for the short sequencing length (usually 20-30 bp) of pyrosequencing (sheend J, et a1.advanced sequencing technologies: Methods and metals, Nat. Rev Genet., 2004, 5(5): 335-44).

Solid-phase pyrophosphate sequencing is a chemiluminescent reaction catalyzed by 3 enzymes, and compared to liquid-phase pyrophosphate sequencing, apyrase is not involved. The solid phase pyrosequencing reaction process is as follows: the DNA template to which the primer is bound is fixed to a support and kept in place during the reaction; after one dNTP is added, the reaction is carried out under the synergistic action of DNA polymerase, ATP sulfurylase and luciferase, and the other reactions are completely the same as liquid-phase pyrophosphate sequencing except that no degradation reaction is carried out; a washing step (washing step) was performed before the next dNTP was added to completely wash away the reaction residue and prevent the accumulation of inhibitory products.

In general, pyrosequencing is referred to as liquid-phase pyrosequencing because its four-enzyme reaction system allows pyrosequencing to be conveniently performed in a single tube.

Ronaghi et al improved the signal-to-noise ratio of pyrosequencing by replacing dATP with dATP. alpha.S (Ronaghi M. et. a1.real-time DNA sequencing using detection of PPi release; anal. biochem, 1996, 242(1): 84-89). Because dATP. alpha.S can be more efficiently utilized by DNA polymerases than dATP, it is more advantageous to read T-rich regions. dATP α S is a mixture of two isomers Sp-dATP α S and Rp-dATP α S, and only Sp-dATP α S can be utilized by the polymerase. In order to obtain the optimum reaction efficiency, it is necessary to maintain the optimum concentration of Sp-dATP α S in the reaction system, and at the same time, the concentration of Rp-dATP α S is also increased. The dATP alpha S still generates an inhibitor of luciferase after being degraded by the Apyrase, so the addition of the dATP alpha S does not improve the reading capability.

The improvement of the method is that only pure Sp-dATP alphaS is added into the reaction, and useless Rp-dATP alphaS is not added, so that the reaction efficiency is improved, the concentration of an inhibition product is greatly reduced, the luciferase can maintain the activity for a longer time, the sequencing length of a pyrosequencing method is increased to 50 bp-100 bp, and the increase of the sequencing length also enables a plurality of new applications of a pyrosequencing technology (Gharizadeh B.et al, Long-reading pyrosequencing using pure 2 ' -deoxysylosine-5 ' -0 ' - (1-thiophosphite) Sp-isomer; Anal Binchem, 2002.301: 82-90).

At the twelfth Conference on Genome Sequencing and Analysis (12th International Genome Sequencing and Analysis Conference) held in 2000, Ronaghi et al proposed a method to remove inhibitory products and reduce dilution effects, increasing the Sequencing length to 200 bp.

Compared with a sanger dideoxy chain termination sequencing method, the pyrosequencing method has the characteristics of rapidness, accuracy, economy and real-time detection; the method does not need gel electrophoresis, does not need any special form of marking and dyeing on a DNA sample, and has high repeatability; a high degree of parallelism and a high degree of automation can be achieved.

Second, progress in application of pyrosequencing technology

1. Application in research of single nucleotide polymorphism

Single Nucleotide Polymorphisms (SNPs) are the third generation of genetic markers that have emerged in recent years and refer to the presence of two different bases at specific Nucleotide positions within the genome, the least of which is at a population frequency of no less than 1%. SNPs are the most common genetic polymorphisms in the genome of an organism and provide a series of markers within or near any one of the genes to be studied; it is this polymorphism in the genome, i.e., differences in genomic sequence, that underlies the genetics of different individuals and populations' susceptibility to disease, and their responses to drugs and environmental factors.

The research of SNP mainly includes two aspects: the method is characterized in that a SNP database is constructed, and all or part of SNPs of a genome of a specific species of organism are mainly discovered. Secondly, the function research of SNP, that is, the SNP is found as the first step of the SNP research, and the research of SNP function is the purpose of the SNP research. Sanger sequencing technology has become a mainstream technology for large-scale, accurate and rapid discovery of SNP. And the pyrosequencing technology which is good at sequencing and verifying short sequences is a good choice when the sequence verification analysis and the frequency analysis are carried out on the existing SNP in the database, and the pyrosequencing technology is adopted for carrying out SNP research, so that the time can be saved and the consumption can be reduced.

Nordfors and the like respectively adopt a Taqman fluorescence quantitative method and a pyrosequencing technology to carry out SNP genotyping research on 1022 samples to obtain the same result, and the control experiment shows that the pyrosequencing technology is a method for carrying out high-efficiency and high-accuracy SNPs of high-throughput and large samples. Wasson et al used pyrosequencing technology to perform SNP allele frequency analysis of DNA pools (DNA pools). Rickert et al, who adopts pyrosequencing technology to perform genotype research on 4-ploid potatoes, can identify 76 allelic loci by pyrosequencing technology in 94 polymorphic loci detection, and the effective rate reaches 81%. Jiangxi and the like use a pyrosequencing technology to identify the haplotypes of the porcine mitochondrial cytochrome b genes. The HLA-DRB genotype analysis research is carried out by utilizing pyrosequencing technology such as Yuanjialin, experiments show that the pyrosequencing result can be accurately genotyped after being compared with the gene sequence of an HLA database, and the method can be applied to the donor/receptor screening of clinical organ transplantation.

2. Application in rapid identification of pathogenic microorganisms

Jonasson et al rapidly identified antibiotic resistance in clinical specimens by detecting the 16S rRNA gene of pathogenic bacteria using pyrosequencing technology. Monstein and the like successfully detect variable V1 and V3 sequences of helicobacter pylori 16S rRNA genes by a pyrosequencing technology, and prove that the technology can meet the requirement of rapid identification and typing of clinical pathogenic bacteria specimens. The Unnerstad and the like use the technology to classify 106 strains of different serotype Listeria monocytogenes, use the pyrosequencing technology to complete a large amount of sample sequencing in a short time, and have very obvious parallelism and high efficiency. The identification and typing of 67 human papillomavirus samples by the technology of Gharizadeh et al prove that the technology is also very suitable for the large-scale identification, typing and mutation research of pathogens such as HPV. Chengshaohui et al extracts viral RNA from Vero-6 cells infected with SARS virus, and uses pyrosequencing technology to sequence multiple base mutation sites and analyze mutation frequency. Through sequencing and analyzing a plurality of possible mutation sites, the virus is determined to be a Beijing epidemic strain.

3. Application in etiology research

Kittles et al analyzed the polymorphism of the CYPl7 gene in three different populations of Nigerian, European and African-American using pyrosequencing technology to study the relationship and clinical manifestations of the CYPl7 gene polymorphism in African-American and prostate cancer. Research results show that the African American people with the CYPl7 genotype of the sequence CC have higher probability of suffering from the prostate cancer than the African American people with the CYPl7 genotype of the sequence TT, and prove that the CYPl7 gene polymorphism of the base C in the population is closely related to the incidence rate of the prostate cancer and is a high risk population. Numerous clues suggest that the COMT gene located on chromosome 22q11 is of major relevance for the pathogenesis of schizophrenia, and that research work by scientists has not yet provided strong evidence; shifman et al proposed an effective method for confirming a high correlation between the occurrence of schizophrenia and the CMOT gene by performing a single nucleotide polymorphism analysis on a large sample of jewish population of the german line using a pyrosequencing technique. The method can also be applied to gene analysis research of other diseases.

4. Application in forensic identification

The Sanger sequencing method for mutation analysis of mitochondrial DNA (mtDNA) cannot achieve accurate quantitative analysis of mtDNA mixtures consisting of contaminants, multiple individual DNAs, etc., whereas Andreasson et al propose a novel quantitative method based on pyrosequencing technology for mtDNA mixture analysis, which can rapidly and accurately detect major and minor mtDNA components from forensic specimen mixed samples. Balitzki-Korte utilizes pyrosequencing technology to perform sequencing analysis on mitochondrial 12SrRNA gene, detects a gene fragment with the length of 149bp, and by referring to a database sequence, can fully determine the biological origin of a subject, for example, whether a piece of skin tissue is from a missing person or an animal.

Third, pyrophosphoric acid analysis device and development

The application of the pyrosequencing technology depends on the research and development of a pyroanalysis device. Regardless of the type of pyrophosphate analysis device, the main structure thereof should comprise two parts: a reactor section and a weak light detection section. The reactor provides a place for reaction, and the weak light detection part is responsible for detecting visible light emitted by the reaction. In the research and application process of the pyrophosphate sequencing technology, reactors designed and used can be mainly classified into 3 types: a microplate reactor, a microfluidic chip reactor and a microarray chip reactor.

Commercial pyrophosphate sequencers are available abroad, but the research reports of related instruments per se in China are few, and no corresponding products are available. A typical representative of foreign products is PSQ96 from Pyrosequencing AB, which was introduced by the company 2001, the system can simultaneously perform independent sequencing of 96 or 384 DNA samples, the time is generally 1 hour and 45 minutes when the sequencing length does not exceed 300bp, the accuracy and reliability reach 99%, and the method has the advantages of high throughput, rapidness and economy. The PSQ96 system has been widely used in basic medical research and clinical molecular diagnostics.

Another representative of the foreign instrumentation study is Genome sequence 20(GS20) introduced in 2005 by 454Life Science, USA. The micro-filtration chamber is used as a reaction environment for pyrosequencing reaction by using MEMS technology, millions of reaction arrays are integrated into an area of 7cm multiplied by 8cm, each reaction chamber can independently and simultaneously carry out sequencing cascade reaction, and a CCD (charge coupled device) with high sensitivity and resolution of an instrument can capture weak fluorescence signals generated by each single reaction chamber, so that sequence information of each sample DNA can be finally obtained. GS20 can realize high-density sequencing reaction only in 4.5 hours, and sequence information of each specimen is obtained through parallel calculation. The method has the advantages of saving the consumption of reaction reagents, reducing the sequencing cost and providing possibility for large-scale sequencing of the genome.

At present, domestic instrument research is just started, a domestic road is long, and the problems are faced in different aspects.

Fourth, importance of sample adding system in pyrosequencing

The pyrosequencing technology and the products thereof provide a very ideal technical operation platform for carrying out timely, rapid and intuitive single nucleotide polymorphism research and clinical examination with large flux and low cost, and are powerful tools for carrying out gene sequence analysis research in the post-genome era. Pyrosequencing technology is being accepted and adopted by more and more researchers, and with the rise of international pyrosequencing technology application and the development of commercial pyrosequencing instruments, the application of pyrosequencing technology in China is rising. However, at the present stage, the application and popularization of the domestic pyrosequencing technology have several limiting factors: (1) the existing commercial pyrosequencing instruments such as PSQ96 and GS20 are expensive; (2) commercial pyrosequencing services are long waiting times and inconvenient; (3) although some laboratories are currently researching devices such as pyrophosphate sequencing chips, and some laboratories have homemade pyrophosphate sequencing test devices with simple structures, the low-end, low-price and commercialized sequence detection instrument based on pyrophosphate sequencing technology is not available in China, and the device is a key problem for restricting the application and development of pyrophosphate sequencing technology.

Pyrophosphoric acid sequencing system is carried out in a micro-environment, the reaction system is usually only 50. mu.L, and the amount of reagents such as reaction substrates, DNA templates, deoxynucleotides and the like required is very small; meanwhile, the sustainability of the cyclic reaction is directly influenced by the amount of single sample addition, and the volume of the reaction solution is rapidly increased by the excessive amount of single sample addition, so that the concentration of the template is reduced too fast. Due to the non-linear increase in reaction delay caused by diffusion, the resulting weak fluorescent signal extends on the time axis and decreases in intensity on the ordinate, resulting in a severe shortening of the nucleic acid's sequenceable length. It is generally considered that the reaction system increase due to the subsequent sample addition is within 10%, and the effect on the experimental results is within an acceptable range. If a 20bp DNA fragment is to be sequenced, the single loading allowed in a 50. mu.L reaction system can be no more than 0.3. mu.L.

In addition to the sample application accuracy, the time accuracy of the sample application interval is also important. Only by adding the dNTPs required for a single cycle at equal time intervals can the degradation of the remaining dNTPs be equivalent for each time, and the effect on the next reaction will be equivalent. The equal time period can provide a reference for the signal of each period, so that the automatic analysis of the nucleotide binding number according to the fluorescence signal intensity is convenient to calculate.

Although the sample adding equipment is relatively abundant, the existing micro sample adding devices in China have defects. For example, the sample adding platform of Shanghai Sunday can be used as a large-scale automatic sample pool processing device to perform sample adding, oscillation and cleaning of a standard 96-well plate, but due to the limitation of a nozzle processing technology, the sample adding micro-precision of the system is only 1 mu L at minimum, and the nL grade required by pyrosequencing cannot be met. After investigation, the method is limited to domestic application and manufacturing level, and the sample adding device in China cannot meet the high requirements on sample adding amount and repetition precision in pyrosequencing.

The university of southeast kudzu vine health badge discloses a liquid phase pyrophosphoric acid analysis device which takes a weak light detection module and a trace dNTP sample adding module as key modules, wherein an air pressure-controlled trace dNTP sample adding module is disclosed, simultaneous sample adding of 96 paths of dNTP solutions can be realized, the minimum sample adding amount is 1.2 mu L, and the maximum error is 13%; however, the signal noise is large, and further improvement is still needed (ge jia hui et al, development of a gene detection device based on pyrosequencing, southeast university, master's academic thesis, 2006).

Wangchunlin and the like disclose a micro sample adding system in a pyrophosphoric acid nucleic acid sequencer by adopting a piezoelectric ceramic nozzle, wherein the sample adding system can be used for alternately adding 4 dNTP reagents to a 96-hole standard plate sample under the driving of a stepping motor, the sample adding repetition precision is more than 95 percent, and the minimum quantity of single sample adding can reach 0.L mu.L. The structure of a trace sample adding system used in the two better types of pyrophosphate nucleic acid sequencers is complex, a sample adding needle is easy to block, dNTP is sprayed into sequencing reaction liquid in different modes and does not contact with the reaction liquid, so that the dNTP is not fully mixed with the reaction liquid and reacts incompletely, the requirement on the dNTP is high, and data is easy to be inaccurate; in addition, the disassembly and assembly are troublesome, the cost is high, and the application under special conditions is not facilitated.

Pyrosequencing (presequencing) is a new DNA sequence analysis technology developed in recent years, which triggers an enzyme cascade reaction by pyrophosphate released after binding of nucleotides and a template, causing fluorescein to emit light and be detected. The technology is an ideal genetic analysis technology platform, can perform DNA sequence analysis, Single Nucleotide Polymorphism (SNP) detection based on sequence analysis, allele frequency determination and the like, and is widely applied to various fields of medical biology and the like at present.

Pyrosequencing is an enzyme cascade chemiluminescence reaction of the same reaction system catalyzed by 4 enzymes, namely DNA polymerase (DNA polymerase), adenosine triphosphate sulfurylase (ATP sulfurylase), luciferase (luciferase) and biphosphatase (apyrase), and the reaction substrates are 5' -phosphosulfate (APS) and fluorescein. The reaction system also comprises a DNA single strand to be sequenced and a sequencing primer. In each sequencing run, 1 dNTP is added, and if it is paired with a template, the polymerase will incorporate it into the primer strand and release an equimolar amount of pyrophosphate groups (PPi). The sulfurylase catalyzes the ASP and PPi to form ATP, which drives the luciferase-mediated conversion of luciferin to oxyluciferin, giving a visible signal proportional to the amount of ATP, and is detected by Pyrogram^TMIs converted into a peak having a height which is positive to the number of nucleotides incorporated in the reactionAnd (4) the ratio. The nucleotide sequence of the template DNA can be recorded in real time based on the type of dNTP added and the intensity of the fluorescent signal. Adenosine triphosphate (dATP) was replaced during the experiment with a-sulfurized adenosine triphosphate (dATP α S) to be efficiently utilized by DNA polymerase without being recognized by luciferin. Since SpdATP α S can reduce the concentration of dATP α S degradation products, in recent years, the problem that single-stranded DNA binding protein (SSBP) and purified Spisomer dATPaS degradation products inhibit the activity of bisphosphatase is better solved, so that the sequencing length can reach 10bp, and the application range of the technology in the field of genetics is expanded.

In pyrosequencing, the DNA sequence is determined by detecting luminescence using a stepwise complementary strand synthesis reaction and a chemiluminescent reaction. The reaction vessel in which complementary strand synthesis is carried out by moving a reaction solution containing pyrophosphoric acid produced by complementary strand synthesis and excess nucleic acid substrates to another reaction vessel to carry out a luminescence reaction, and decomposing the excess nucleic acid substrates through a region to which an enzyme decomposing the excess nucleic acid substrates is immobilized while the reaction solution is moving, and then converting pyrophosphoric acid into ATP to be introduced into a chemiluminescent reaction vessel. However, the prior art has the disadvantages that a large amount of substrates and enzymes must be added for reaction to ensure that the reaction can be completely carried out, and then the reaction is carried out after excessive substrates are removed, so that the reaction process is increased, the cleaning and reaction difficulty of each step is increased, reagents such as the substrates and the like are seriously wasted, the reaction time is long, manpower and material resources are wasted, and the popularization and use potential of the pyrosequencing in the market is invisibly reduced.

Most of the existing instruments applied to pyrosequencing are monopolized by part of manufacturers for manufacturing and selling, the instruments and reagents are sold in a matched mode, the cost is very high during sequencing, the detection and maintenance process is dependent on specific technicians, the period is long, the cost is high, the volume required by reaction is large, the reaction cost is increased, the detection result is unstable, and the repetition precision is low. Therefore, it is necessary to design a DNA sequencer suitable for pyrosequencing.

Fifth, general description of DNA Single-Strand separation technique

The DNA single-strand separation technology is one of the most common separation technologies in the field of biomedicine, is suitable for DNA sequencing and probe equipment of different nucleic acid samples in various scales, and is widely applied to the fields of biology, medicine and pharmacology, preventive medicine, animal and plant science, agriculture and animal husbandry, food and hygiene, energy and chemical industry, environmental monitoring, medical diagnosis and detection and the like. In addition, the techniques of adsorption, extraction and separation of DNA single strands are widely used in water quality, water sources, biological materials, biological fluids (such as blood, serum, plasma, cerebrospinal fluid, urine, tears, sweat, digestive juice, semen, secretion, interstitial fluid, vomit, stool), tissue/cell and microorganism lysates, analysis, separation and purification of biological, chemical molecules and drugs such as proteins, nucleic acids and the like from different sources, and synthesis of oligonucleotides, polypeptides, lead compounds and drugs.

The DNA single-strand separation method commonly used in the biomedical field has the following disadvantages:

1. heat denaturation or alkali treatment. The method mainly comprises the step of heating or alkali treatment of a double-stranded PCR product, and the DNA double strands are broken by hydrogen bonds under a high-temperature or certain-degree alkaline environment, so that the DNA is changed into single strands. Although the principle of the method is feasible and the operation is simple, the method is not used for purifying single-stranded DNA gradually due to low separation rate and purity, and is mostly used for double-stranded separation of DNA.

T7 reverse transcription method. The method is to add a T7 promoter at the 5' end of a PCR primer, use the purified PCR amplification product as a template, and synthesize single-stranded RNA by T7RNA polymerase in vitro reverse transcription (Hughes, et al, Nat. Biotechnol.,2001,19: 342-one 347). Although the principle of the method is feasible, the separation rate of the DNA single strand is high, and the purity of the obtained DNA single strand is high, the whole separation process needs to be completed by two steps, so the method is inconvenient to operate and long in time, and the pollution of the RNA enzyme needs to be strictly controlled, so the method has certain limitations.

3. Exonuclease methods (Higuchi and Ochman, nucleic acids Res.,1989,17: 5865). Since one PCR primer is phosphorylated, the phosphorylated primer amplified strand is not cleaved when the PCR product is digested with exonuclease, and the enzyme is heat inactivated after digestion. The method also needs to purify PCR products, has long separation procedure and very inconvenient operation, and the DNA single-strand yield depends on the activity of exonuclease, the uncontrollable factor is too strong, and the stability of the experimental result is not enough; therefore, the method has a low implementation rate and low versatility.

4. Denaturing high-performance liquid chromatography (DHPLC). Under partially denaturing conditions, DNA mutations are found by the difference in retention time in the column between heterozygous and homozygous diploids. The melting properties of the heteroduplex and the homoduplex are different, under partial denaturation conditions, the heteroduplex is more easily denatured due to the presence of mismatched regions, and the retention time in the chromatographic column is shorter than that of the homoduplex, so that the heteroduplex is eluted first and appears as a bimodal or multimodal elution profile in the chromatogram. Since one PCR primer is labeled with biotin, the PCR-amplified strand will be separated from the other common strand in DHPLC (Dickman and Hornby, anal. biochem.,2000,284: 164-. The method can directly obtain the required DNA single strand from the double-stranded PCR product within 15min, but the implementation of the method needs to be matched with a very expensive instrument, so the method is difficult to popularize all the time.

5. Magnetic bead capture method. The surface of the superparamagnetic nano-particles is improved and modified by using a nanotechnology to prepare superparamagnetic silicon oxide nano-magnetic beads. The magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface. By utilizing the superparamagnetism of the silicon oxide nano microspheres, DNA and RNA in samples such as blood, animal tissues, food, pathogenic microorganisms and the like can be separated under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate and the like) and an external magnetic field, and then a target single chain is obtained by treating with NaOH. The method is simple to operate and short in time consumption, the whole extraction process is divided into four steps, most of the four steps can be completed within 36-40 minutes, the method is safe and non-toxic, toxic reagents such as benzene and chloroform in the traditional method are not used, harm to experiment operators is reduced, the modern environmental protection concept is met, the extracted DNA single strand is high in purity and high in concentration due to the specific combination of the magnetic beads and the DNA single strand, but the coated magnetic beads used in the method are expensive and need to be separated by a magnetic frame, the separation cost is high, and the method is inconvenient, so that the popularization of the technology is limited to a certain extent.

6. Asymmetric PCR. The above methods all require additional processing after PCR, and asymmetric PCR can prepare single DNA strands while PCR amplification is performed. Conventional asymmetric PCR uses two unequal amounts of primers for normal amplification in the initial cycle. As the cycles increased, the smaller amount of primers were gradually depleted, while the excess primers continued to amplify linearly to form DNA single strands (Gyllensten and Erlich, Proc. Natl. Acad. Sci. U.S.A.,1988,85: 7652-7656). The method has higher hybridization sensitivity and specificity and stronger operation simplicity, but the proportion of the primer needs to be optimized, the chance of nonspecific amplification is increased, in addition, the DNA single-strand separation process needs to depend on electrophoresis, the separation procedure is complicated, and the electrophoresis often can see a dispersed band, so that the time consumption and inconvenience are obvious.

The separation methods have certain limitations, therefore, in order to meet the requirements of operability and economy of DNA single-strand separation, the DNA single-strand in the prior art adopts an integrated extraction workstation, an affinity connector with streptavidin is combined with the DNA double-strand, the workstation is provided with a suction filtration needle and a matched pump, the combined DNA affinity connector is adsorbed on the lower part of an inner filter membrane of the suction filtration needle through suction filtration, the workstation is provided with a track and a related system, the suction filtration needle is moved to a disc filled with NaOH after the suction filtration is finished, double helix decomposition is carried out through alkali treatment, a DNA single-strand is obtained, and the DNA single-strand is cleaned and collected after the suction filtration is finished again. Generally, 24 (4 x 6) suction filtration needles are in a group, and a sufficient amount of samples or reagents must be guaranteed to guarantee the normal operation of a workstation during use, so that the collection mode of the DNA single strand is quite inflexible, the DNA single strand can only be added into the workstation in a fixed amount to work, a large amount of loss is generated in the processes of multiple suction filtration and transfer, the collection of trace amount is quite unfavorable, and the suction filtration needle group needs to work simultaneously, the suction filtration needle group has certain volume requirements on all parts of the workstation, and the whole workstation occupies a large space. The huge system causes that in the operation process of DNA single-strand separation, the micro-separation column needs to repeatedly transfer liquid, the operation is very complicated, the separation period is long, the efficiency is low, the whole equipment is expensive, the cost of DNA single-strand separation is high, a large amount of reagents and other resources are required to be consumed, and the operation is extremely uneconomical. In addition, the suction needle in the workstation is made of metal, is expensive, is often reused after treatment, is easy to cause cross contamination between residues, has low reliability, and causes certain interference and influence on the accuracy of separation and detection results. And in the solution extraction process, part of residual solution adheres to the wall, so that a certain amount of target DNA single chains cannot be adsorbed by the micro separation column, the proportion of the obtained DNA single chains is reduced, the separation rate is influenced, and waste is caused. Therefore, the problem of high-quality and high-efficiency DNA single-strand separation for pyrosequencing is urgently solved.

Disclosure of Invention

In view of the above problems in the prior art, the present invention provides a DNA sequencing apparatus based on pyrophosphate sequencing, which is easy and fast to operate.

In order to achieve the purpose, the invention adopts the following technical scheme:

a DNA sequencing device based on pyrosequencing comprises a sample area, a reaction area and a detection 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 frame, dNTP reagent tanks and a reaction tank, a plurality of sample adding needles are fixedly arranged on the sample adding frame, the sample adding frame reciprocates between the dNTP reagent tanks and the reaction tank through a linear moving device, a rotating shaft is arranged in the center of the sample adding frame, the rotating shaft drives the sample adding frame to rotate through a motor, a lifting device is arranged between the sample adding frame and the rotating shaft, and the sample adding frame is lifted along the rotating shaft through the lifting device; the reaction tank is provided with a plurality of reaction sites, and the reaction sites extend to a detection zone;

the detection area comprises a bioluminescence detector and a rotating platform, the bioluminescence detector is detachably arranged on the rotating platform, and the rotating platform drives the bioluminescence detector to rotate through a motor; the sample area, the reaction area and the detection area are arranged on the analysis table, a manipulator for operation is arranged on the side face of the analysis table, and the linear moving device is fixed on the analysis table through a mounting frame.

As a further improvement of the above technical solution:

preferably, the upper end of pivot is equipped with the fixing base, be equipped with the bearing between fixing base and the pivot.

Further, the linear moving device comprises a movable slide rail fixed on the mounting frame and a movable slide block fixed on the fixing seat.

Furthermore, the lifting device comprises a lifting slide rail fixed on the rotating shaft and a lifting slide block fixed on the sample adding frame.

Preferably, the filter membrane is provided at one end of the filter column. In other words, the filter membrane forms the bottom end of the filter column, and the filtration surface of the DNA isolation zone is arranged at the bottom of the separation column, i.e.the single-stranded DNA after separation is on the filter membrane.

Further, as a preferred embodiment, the outer diameter of the filter column is equal to the inner diameter of the collecting pipe, so that the filter column and the collecting pipe can be kept in a relatively stable state through friction force, and an additional structure is not needed for limiting and fixing the filter column.

In another preferred form, the separation discs are provided with a plurality of collection tubes in which the filtration columns are mounted, the filter membranes being located at the non-end of the filtration columns.

Furthermore, the filter membrane is in a polymer nano microsphere structure, and the aperture among the polymer nano microspheres is smaller than the diameter of the affinity connector.

Furthermore, the collecting pipe is provided with a detachable upper cover, and the upper cover is connected with the collecting pipe through a connecting belt.

The separated single-stranded DNA is a single strand connected with an affinity body on a filter membrane, the single strand is required to be capable of eluting the filter membrane, the filtrate contains another complementary strand without the affinity connector, and the single-stranded DNA in the filtrate can be collected when reverse sequencing is required. When the chain of taking the affinity connector on the filter membrane needs to be collected, the collecting pipe can adopt the collecting pipe of retrieving, and the collecting pipe only plays the effect of retrieving the waste liquid this moment, recycles reduce cost to the production of white pollution is reduced in the environmental protection.

The sample adding frame is circular, the sample adding needles are uniformly distributed along the circumferential direction, and the reaction positions are uniformly distributed on the reaction tank along the circumference with the same diameter as the sample adding needles.

The reaction sites are made of transparent materials, and the bioluminescence detector is positioned in a circle surrounded by all the reaction sites.

The bioluminescence detector is a CCD camera, and a clamping groove for placing the CCD camera is arranged on the rotating platform.

The reaction zone further comprises a cleaning tank and a drying tank which are arranged on the analysis table, and the cleaning tank and the drying reaction are positioned between the dNTP reagent tank and the reaction tank.

In view of the above problems in the prior art, the DNA sequencing device based on pyrophosphate sequencing according to the present invention has the following advantages:

(1) the DNA single strand is extracted in a filtration and centrifugation mode, so that economical and miniaturized collection of a sample is realized, and the high-efficiency low-loss rapid DNA single strand separation method and device are provided. The method is simple and easy to operate, the time for obtaining the target sample is short, the efficiency is high, the method can be used for collecting and extracting trace DNA single strands, the sample loss can be almost ignored, the used reagent is less, the requirement on equipment is low, a water pump is not needed in the separation process, the equipment configuration is greatly simplified, the total equipment cost is reduced, the operation steps are effectively simplified, the operation time is shortened, the working intensity is reduced, and the working efficiency is improved; the filter column matched with the conventional centrifuge tube is adopted, so that the separation quality and efficiency are ensured, the separation process is simple and easy to realize, and the filter membrane of the filter column separates the DNA single chains in a physical mode, so that the separation difficulty and condition requirements are reduced. The filter membrane is made of homogeneous materials with the same structure, can be exchanged in two directions for use, increases the design possibility of the filter column, can be made into a hollow structure with the filter membrane at one end, enriches the structure and the variety of the DNA single-strand separation device, increases the application occasions of the DNA single-strand separation device of the invention on the premise of ensuring the same function, avoids the single structure of the product, and obviously enhances the adaptability; and the structure can be vertically symmetrical and can be used in a vertically-replaceable and matched way. This DNA single strand separator adopts low price's PC plastics, and economy and practicality, the design of infundibulate inner chamber is favorable to the gathering and the guide of reaction liquid for reaction liquid separation is more abundant more complete, has obtained higher proportion purpose DNA single strand, has guaranteed higher separation rate, has avoided extravagant. In addition, the DNA single-chain separation device is also suitable for a commercially available centrifuge tube and is designed in a standardized manner, so that the DNA single-chain separation device is extremely strong in universality and extremely wide in application range, and therefore, the application prospect is very wide.

(2) The sample adding method and the device are suitable for any detection device, are simple and convenient to disassemble, are simple and convenient to clean, cannot cause the defects of needle blockage and other sample adding devices in the prior art, have sample adding repetition precision of more than 95 percent, have the minimum sample adding amount of 0.1 mu L, have good sample adding uniformity, shorten the sequencing time greatly and have extremely high result accuracy; in addition, the nucleic acid sequence of the sample can be qualitatively and quantitatively detected by the combined detection device; therefore, the application prospect is very wide.

(3) The DNA sequencing device based on the pyrosequencing provided by the invention adopts the manipulator to control the whole process, has no infection and high accuracy, and improves the efficiency and precision.

In a word, the DNA sequencing device based on pyrosequencing provided by the invention reduces the usage amount of substrates and enzyme systems, has accurate detection, high reaction speed and high integration, can simultaneously detect one or more SNP sites and single-stranded DNA fragments, breaks monopoly of part manufacturers for the enzyme systems and the substrates, greatly reduces the price, has precise structure of an analysis device, has low requirement on the usage amount of the DNA fragments and the substrates to be detected, reduces the detection cost of pyrosequencing, and enlarges the application range of the pyrosequencing.

Drawings

FIG. 1 is a schematic diagram showing the structure of a DNA sequencing apparatus based on pyrophosphate sequencing according to the present invention (separation tray not shown).

Fig. 2 is a schematic top view of the structure of fig. 1.

FIG. 3 is a preferred embodiment of the present invention for use in DNA isolation regions of pyrophosphate.

FIG. 4 is a schematic structural view of a filtration column in example 1 of the present invention.

FIG. 5 is a schematic structural view of a filtration column in example 2 of the present invention.

FIG. 6 is a schematic structural view of a filter column in example 3 of the present invention.

FIG. 7 is a schematic structural view of a filtration column in example 4 of the present invention.

FIG. 8 is a schematic view of a sample application needle in a sample application device structure for a pyrophosphate sequencing system according to the present invention.

The reference numbers in the figures illustrate:

1. an analysis stage; 11. a mounting frame; 2. a separation disc; 21. a filtration column; 211. a separation channel; 22. filtering the membrane; 23. a collection pipe; 231. an upper cover; 232. a connecting belt; 3. a reaction tank; 31. reaction sites; 4. a sample adding frame; 41. a sample adding needle; 42. a rotating shaft; 43. moving the slide block; 44. a fixed seat; 45. moving the slide rail; 46. lifting the slide rail; 47. a lifting slide block; 5. a bioluminescent detector; 51. a rotating table; 511. a card slot; 6. a drying tank; 7. a dNTP reagent reservoir; 71. a slot position; 8. and (4) cleaning the tank.

Detailed Description

The present invention will be described more fully hereinafter with reference to the following examples.

Example 1

FIGS. 1 to 4 show a first embodiment of a DNA sequencing apparatus based on pyrosequencing according to the present invention. The DNA sequencing device based on pyrosequencing can be used for pyrosequencing detection and analysis of DNA sequences, the DNA sequence to be sequenced is a target sequence, and pyrosequencing is carried out after the target DNA sequence is amplified. The DNA sequencing apparatus of this embodiment includes a sample zone, a reaction zone, and a detection zone. The sample zone, the reaction zone and the detection zone are mounted on the analysis table 1, and the sample zone, the reaction zone and the detection zone are monitored and controlled by the control zone. The whole analysis process adopts manipulator operation.

Before pyrosequencing, the sequenced DNA template needs to be amplified to reach the DNA concentration required by amplification. When the amplification primer is designed, the amplification primer is provided with an affinity connector, the affinity connector is preferably biotin, the affinity connector is preferably marked on one end of the primer of the target DNA, and PCR amplification can be carried out by adopting the prior art.

The sample section comprises separation discs 2, which separation discs 2 are placed in a centrifuge. After the target DNA is amplified, the target DNA is double-stranded DNA, single-stranded DNA is needed for pyrosequencing, the separation disc 2 comprises at least one DNA separation area, the DNA separation area adopts a physical filtration mode, the separation area comprises a hollow filtration column 21 with a filtration membrane 22 arranged inside, the filtration membrane 22 is in a high-molecular nanometer microsphere structure, as the aperture among the high-molecular nanometer microspheres is smaller than the diameter of an affinity connector, the single-stranded DNA marked by the affinity connector is remained on the membrane, and the single-stranded DNA with the mark or a complementary strand thereof is collected according to sequencing needs and is used for pyrosequencing.

In this embodiment the separation disc 2 is provided with a plurality of collection tubes 23, the filter column 21 being mounted in the collection tubes 23, the filter membrane 22 being located at the end of one end of the filter column 21. The outer diameter of the lower section of the filter column 21 is the same as the inner diameter of the collection pipe 23.

After alkaline hydrolysis, one strand of the double-stranded DNA to be subjected to affinity connection is left on the filter membrane 22, and the strand is collected only by adding a collecting solution into the filter column 21; if the complementary strand is collected, the filtrate can be collected, and then the complementary strand in the filtrate can be collected.

As shown in fig. 3, the collecting tube 23 is used for collecting waste liquid generated during centrifugation, and needs to be poured in time after each step to prevent cross contamination, and the upper end tube body of the collecting tube 23 is a cylinder, the lower end is a cone, and the bottom is spherical. In another preferred embodiment, the collection pipe 23 is provided with an upper cover 231, the upper cover 231 is detachably connected to the collection pipe 23 by a connecting band 232, the upper cover 231 can be tightly buckled on the filter column 21 or the collection pipe 23 after the filter column 21 is installed due to the connection of the connecting band 232, and the upper cover 231 is used for preventing the liquid from splashing out of the filter column 21 to cause loss and pollution during the liquid centrifugation.

The preferable filter membrane 22 material in this embodiment is polyethylene microspheres, the pore space between the microspheres is preferably 10 μm, which is smaller than the diameter of the affinity connector, the single chain with the affinity connector can be directly left on the membrane by physical filtration, and the single chain not connected with the affinity connector is filtered out, so that the adsorption and elution effect is good, the DNA recovery rate is high, and the raw material price is low and the environment is protected.

As shown in fig. 4, in order that the filter membrane 22 in this embodiment does not move during the blowing or centrifuging process, it is also preferable in this embodiment to provide a film pressing device above the filter membrane 22, the film pressing device including a gasket and/or a film pressing frame. The liquid to be separated and purified passes through the pad and then contacts the filter membrane 22, and the pad is preferably made of fiber materials, can resist acid, alkali and most organic solvents, and cannot adsorb most biological molecules.

Preferably, a membrane pressing frame is arranged above the gasket and on the side not in contact with the filter membrane 22, the membrane pressing frame is made of the same material as the filter column 21, and the filter membrane 22 is pressed by mechanical pressure, so that the filter membrane 22 cannot move in the use process of blowing, centrifuging and the like, and the collection loss is caused.

Since the diameter of the affinity linker is larger than the diameter of the filter 22 of the DNA single strand separation apparatus, when the DNA single strand passes through the filter 22, the DNA single strand not bound to the affinity linker and other impurities can pass through the filter 22, while the single strand bound to the affinity linker is left on the membrane and cannot pass through, and the filtration is physical.

The reaction area comprises a sample adding frame 4, a dNTP reagent groove 7, a cleaning groove 8, a drying groove 6 and a reaction groove 3, wherein the cleaning groove 8 and the drying groove 6 are positioned between the dNTP reagent groove 7 and the reaction groove 3.

The dNTP reagent tank 7 is provided with four slots 71, and before the reaction, the substrate supply part feeds different nucleic acid substrates of dNTP to the slots 71 through a pipe. The substrate supplying part is used for supplying dNTPs, and provides an environmental basis for hybridization reaction with target DNA. dNTP used for pyrosequencing comprises four nucleic acid substrates of dATP, dCTP, dGTP and dTTP, wherein the substrates are used for hybridizing with target DNA, and related enzyme systems are required to be added into a reaction system to catalyze the reaction, particularly DNA polymerase. In the complementary strand synthesis reaction, the by-product pyrophosphoric acid obtained during complementary strand synthesis is converted to ATP, and ATP and luciferin are reacted in the presence of luciferase to emit light. Since pyrophosphate is produced when complementary strand synthesis occurs and light is emitted as a result, it is possible to identify the DNA sequence by monitoring the occurrence of complementary strand synthesis, that is, the type of incorporated base.

A plurality of sample adding needles 41 are fixedly arranged on the sample adding frame 4, the sample adding frame 4 is circular, and the sample adding needles 41 are uniformly distributed along the circumferential direction. The sample adding frame 4 reciprocates among the dNTP reagent tank 7, the washing tank 8, the drying tank 6 and the reaction tank 3 through a linear moving device, and the linear moving device is fixed on the analysis table 1 through a mounting frame 11. The center of application of sample frame 4 is equipped with pivot 42, and pivot 42 passes through motor drive application of sample frame 4 and rotates, is equipped with elevating gear between application of sample frame 4 and the pivot 42, and application of sample frame 4 is elevating movement along pivot 42 through elevating gear. The sample addition needle 41 is a solid needle for transferring dNTP reagents, and the sample addition needle 41 is fixed in the through hole of the sample addition frame 4. Transferring the DNA single strand to a reaction site 31 according to the required amount of a reaction system, adding the enzyme system, then sequentially adding dNTP, wherein the adding sequence is not limited, and for each site, adding four substrates once respectively. The upper end of the rotating shaft 42 is provided with a fixed seat 44, a bearing is arranged between the fixed seat 44 and the rotating shaft 42, and the fixed seat 44 does not rotate when the rotating shaft 42 rotates. The linear moving device comprises a moving slide rail 45 fixed on the mounting frame 11 and moving sliders 43 fixed on two sides of the fixed base 44. The lifting device comprises a lifting slide rail 46 fixed on the rotating shaft 42 and a lifting slide block 47 fixed on the sample adding frame 4. The linear moving device and the lifting device adopt linear guide rails.

The reaction tank 3 is provided with a plurality of reaction sites 31, and the reaction sites 31 are made of a transparent material and extend to the detection region. The reaction sites 31 are uniformly distributed on the reaction tank 3 along a circumference having the same diameter as the sample addition pins 41.

The detection area comprises a bioluminescence detector 5 and a rotating platform 51, wherein the bioluminescence detector 5 is a CCD camera and is connected with a computer to display a detected spectrogram. The rotary table 51 is provided with a slot 511 for placing a CCD camera. The CCD camera is located in a circle surrounded by all the reaction sites 31. The rotating platform 51 drives the bioluminescent detector 5 to rotate through a motor.

The DNA sequencing system based on pyrosequencing provided by the invention can better transfer substrates and DNA and reduce loss in the transfer process.

The shape of the reaction region is not limited, and the specific structure in the analysis device can be designed and adjusted according to the detection requirement, and any structure shape and relative position that can realize pyrophosphate sequencing should be considered to fall within the protection scope of the present invention.

Adding a substrate into the reaction area, adding a DNA single strand to be detected and other reagents required in a reaction system into the reaction area, and calculating the amount of 5ul required for detection, wherein the reaction system is as follows:

the enzyme mixture comprises:

the substrate mixture comprises:

APS 0.4mg/L；

firefly luciferin 0.4 mmol/L.

In the reaction process, the optimum pH value for the enzyme activity is selected at each step for reaction, the pH value in the reaction system needs to be adjusted after the reaction to adapt to other reactions, and the reaction conditions in the specific reaction system can be obtained by a person skilled in the art according to the prior art. For example, when apyrase is included in the washing step to remove excess nucleotide species and ATP, because of the continuity of the treatment step, a buffer may be used with apyrase at a pH that optimizes the level of apyrase cleanliness. Then, a polymerase is used in the next nucleotide incorporation step,different buffers with optimal PH conditions for the polymerase can be used in order to optimize polymerase cleanliness. In addition, each optimal buffer may include a preferred counter ion for each enzyme, e.g., Ca for apyrase buffer²⁺And Mg for polymerase buffer²⁺。

The sequencing result is judged by bioluminescence, and other devices capable of detecting bioluminescence can be used as the detection device, which is not limited herein.

The pyrophosphate sequencing-based DNA sequencing device further comprises liquid supply and discharge channels between the sample storage devices, wherein the liquid supply channel is used for conveying reagents and samples to be reacted, and the liquid discharge channel is used for discharging liquid after the reaction or after centrifugation to the collection part.

In the analysis device, during the reaction, the operation of each part needs to be ensured to be carried out under the most suitable environment, the enzyme system needs to be reacted in the reaction system with the highest activity to ensure the reaction to be complete, and therefore, a plurality of components which can ensure the reactions to be carried out efficiently and orderly are arranged in the control area, and the control area comprises a centrifuge, a pH meter, a heating component, a control signal transmission component and other routine structures of sequencing equipment.

The system and method of the described embodiments of the invention may include some design, analysis, or other operations performed using a computer-readable medium having stored thereon instructions for execution on a computer system. For example, some embodiments of processing detected signals and/or analyzing data generated with sequencing results systems and methods, where the processing and analyzing embodiments are performed on a computer system.

An exemplary embodiment of a control area for use with the present invention may include any type of computer platform, such as a workstation, a personal computer, a server, or any other existing or future computer. A computer typically includes well-known components such as a processor, an operating system, system memory, memory storage devices (memory storage devices), input/output controllers, input/output devices, and a display. One of ordinary skill in the relevant art will appreciate that there are many possible computer configurations and components, and may also include high speed memory, data distribution units, and many other devices.

The display may include a display that provides visual information, such information typically being logically and/or physically organized as an array of pixels. An interface controller may also be included, which may include any of various known or future software programs for providing input and output interfaces. For example, the interface may include a so-called "Graphical User Interfaces (commonly referred to as GU I)" that may provide one or more Graphical displays to a User. The interface is generally capable of accepting user input using selection or input means known to those of ordinary skill in the relevant art.

In the same or alternative embodiments, applications on a computer may use an interface that includes a so-called "command line interface" (commonly referred to as a CLI). CLI typically provides text based interactions between applications and users. Typically, a command line interface displays output and receives input in the form of text lines through a display. For example, some implementations may include a so-called "command line interpreter" (Shell), such as the Unix command line interpreter (Unix Shell), known to those of ordinary skill in the relevant art, or Microsoft Windows Powershell, such as the Microsoft.

One of ordinary skill in the relevant art will appreciate that the interface may include one or more GUIs, CLIs, or combinations thereof.

The processor typically executes an operating system, which may be any operating system known in the art, and is considered to be within the scope of the present invention as long as the skilled person can process the detection results and data.

The DNA sequencing device based on the pyrosequencing can be widely used for determining, diagnosing and checking DNA sequences, determining and diagnosing SNP sites and the like, can realize simultaneous sequencing of a certain sample, has low requirement on sequencing conditions on the principle, does not need to additionally add expensive experimental reagents such as an excitation light source and a fluorescent agent, can realize simple and cheap DNA sequencing work, has stable detection result and high accuracy, overcomes the defect that the conventional equipment cannot be fully carried out or complementary strand synthesis reaction is carried out excessively first, has small reaction volume, and can finish the work in a reaction stage in shorter time.

The analysis method adopting the DNA sequencing device and the system based on pyrosequencing provided by the invention comprises the following steps, wherein the undisclosed part can refer to the prior art:

(1) combining: and combining the amplified DNA fragment with the agarose beads, wherein the length of the DNA fragment is 10-20 kb, the minimum sample loading amount of the DNA is not less than 500ng, the diameter of the agarose beads is 30 mu m, the surface of the agarose beads is coated with biotin and streptavidin, and the amplified DNA fragment is spontaneously and specifically combined with the streptavidin-coated agarose beads.

(2) Centrifuging: sucking the combined DNA double chains into a filter column 21, putting the filter column 21 into a collecting pipe 23 in advance, putting the filter column into a centrifuge, centrifuging for 1min at 12000rmp, and removing the redundant solvent; wherein the collecting tube 23 is a 1.5mL centrifuge tube; the filtering column 21 is a cylinder with an opening at the upper end and a semi-closed lower end, a boss is arranged on the outer edge of the opening at the upper end of the filtering column 21 and fixed on the collecting pipe 23, a filtering membrane 22 is arranged in the lower end of the filtering column 21, the diameter of the filtering column 21 is 4.5mm, the diameter of the filtering membrane 22 is 4.7mm, and the filtering membrane 22 is forcibly pressed into the filtering column 21 to be tightly attached without gaps.

(3) Washing: adding a proper amount of 70-80% ethanol, washing off residual amplification reagents such as Taq enzyme and the like, gently blowing, uniformly mixing, and centrifuging at 12000rmp for 1 min.

(4) Alkaline hydrolysis: adding 0.4M NaOH and 1M NaCl to unwind the double-stranded helix, gently blowing, beating and mixing uniformly, and then centrifuging for 1min at 12000 rmp.

(5) Adjusting the pH value: adding a proper amount of elution buffer or ultrapure water for cleaning, washing out residual NaOH, balancing the pH value to be neutral, slightly blowing, uniformly mixing, and centrifuging at 12000rmp for 1 min.

(6) Collecting: adding a collecting solution such as ultrapure water and the like, adding the collecting solution, gently blowing until the DNA single strand is completely suspended, sucking the DNA single strand out for pyrosequencing, adding the sucked DNA single strand into a reaction site 31, and adding the enzyme required for the reaction.

(7) Sample adding: the sample adding frame 4 is moved to the position above the dNTP reagent tank 7, the sample adding frame 4 is lowered, and any one dNTP reagent is dipped by the sample adding needle 41 so that the dNTP reagent is attached to the periphery of the sample adding needle 41.

(8) Reaction: the sample adding frame 4 is moved to the upper part of the reaction tank 3, the sample adding frame 4 is rotated, the sample adding needle 41 dipped with the dNTP reagent is aligned to the reaction site 31 to be tested, the sample adding frame 4 descends, the sample adding needle 41 attached with the dNTP reagent is inserted into the sequencing reaction solution, and then the sample adding needle 41 is separated from the sequencing reaction solution.

Among them, in order to complete sequencing, it is preferable to add 4 kinds of dNTP reagents in any sequence or multiple sequences to be tested into the sequencing reaction solution, for example: when only detecting whether a single base is changed, the position of the base to be detected can be determined according to the known sequence before and after, the process of adding the sample (adding 4 dNTP reagents) for 4 times is repeated in sequence, and the base is added into the same sequencing reaction solution to detect the base.

When the sampling needle 41 leaves the sequencing reaction solution, the sampling frame 4 moves to the cleaning tank 8 to clean the sampling needle 41, dries the cleaned sampling needle 41, and dips in a next dNTP reagent.

(9) And (4) conclusion: the CCD camera is connected with a computer for taking a picture and displaying the detected spectrogram.

The solution, parameters and other separation conditions involved in the DNA separation process in this example are preferred embodiments in this example, and any experimental conditions, parameters and solutions that can perform corresponding functions with reference to the prior art can be used in the separation and purification process in this invention, and the specific parameters and solutions in this example should not be construed as limitations of the present invention.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 96%.

Example 2

Since one strand collected on the filter membrane 22 needs to be sucked out or poured out, and such a collection method may cause a certain collection loss, the inventors preferably set the filter column 21 to have a double-head structure with two ends exchanged for use, and set the filter membrane 22 on the non-end surface of the filter column 21, and when collection is needed, the two ends of the filter column 21 are exchanged and then the whole DNA single strand on the filter membrane 22 can be eluted by a conventional method, such as centrifugation, to reduce the sample loss.

As shown in fig. 5, the only difference between this embodiment and embodiment 1 is that the filter column 21 in embodiment 2 is a hollow cylinder with a vertically symmetric structure, the filter membrane 22 is vertically located in the middle of the hollow cylinder, the filter column 21 can be used by replacing two ends, the inner diameter of the hollow cylinder is 4.5mm, the diameter of the filter membrane 22 is 4.7mm, the filter membrane 22 is forced into the filter column 21 to be tightly attached without gap, and a membrane 22 limiting and pressing mechanism (not shown) is added in the cylinder to fix the filter membrane 22 so as to prevent displacement. Because the two ends of the DNA single-strand separation device are the same in structure and function, the DNA single-strand separation device does not need to distinguish the liquid inlet end from the liquid outlet end, and the two ends can be randomly selected for use when in use.

After the surplus NaOH is washed away in the step (4), two ends of the filter column 21 are exchanged, the collection liquid is added, the filter column is kept still for more than 1min, the elution step can be carried out without blowing, the rotating speed of a centrifugal machine does not exceed 10000rpm during elution, and the filter column is centrifuged for at least 2 min. After collection, the DNA concentration can be detected by nanodrop, and pyrosequencing can be performed.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 95%.

Example 3

As shown in FIG. 6, this embodiment is different from embodiment 1 only in that the filter column 21 of the DNA single strand separation apparatus in embodiment 3 is a spatial circular truncated cone type structure, the filter membrane 22 is disposed on the common top surface of the two circular truncated cone type filter columns 21, the diameter of the top surface of the circular truncated cone type filter column 21 is the same as that of the filter membrane 22, and the filter membrane 22 is provided with a squeeze film mechanism (not shown) on both sides thereof to fix the filter membrane 22 against displacement. The side wall of the lower end of the opening of the circular truncated cone-shaped filter column is provided with a boss, so that the circular truncated cone-shaped filter column can be fixed on the boss of the collecting pipe 23 when the two ends are exchanged.

The separation method using the separation apparatus pertaining to the present embodiment is the same as the procedure described in embodiment 1, differing from embodiment 1 only in that: after the surplus NaOH is washed away in the step (4), two ends of the filter column 21 are exchanged, the collection liquid is added, the filter column is kept still for more than 1min, the elution step can be carried out without blowing, the rotating speed of a centrifugal machine does not exceed 10000rpm during elution, and the filter column is centrifuged for at least 2 min. After collection, the DNA concentration can be detected by nanodrop, and pyrosequencing can be performed.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 95%.

Example 4

As shown in fig. 7, the present embodiment is different from embodiment 1 only in that, in order to better gather and guide the reaction solution, so as to separate the reaction solution more fully and completely, and avoid dead filtering angles, the present embodiment adds a separation channel 211 on the basis of embodiment 1, the separation channel 211 is disposed between and communicated with two filter columns 21, the two filter columns 21 and the separation channel 211 share a central axis, the filter membrane 22 is disposed on a symmetrical plane of the separation channel 211, two sides of the filter membrane 22 are provided with a squeeze film mechanism (not shown) to fix the filter membrane 22 against displacement, and the middle portions of the two filter columns 21 are provided with a boss to ensure that the two ends can be fixed on the boss of the collecting pipe 23 when being exchanged.

The one end that filter column 21 and separation channel 211 are connected is inverted circular truncated cone form, and the two constitutes a hourglass hopper-shaped spatial structure jointly, and the bottom surface diameter of inverted circular truncated cone end is unanimous with filter column 21 internal diameter, and the top surface diameter is unanimous with separation channel 211's diameter, and the structure be provided with the gathering and the guide that do benefit to reaction liquid for reaction liquid separation is more abundant more complete. Therefore, the DNA single-strand separation device in this embodiment enables the DNA single-strand filtration and separation to be more thorough on the basis of embodiment 2, and the separation efficiency is significantly improved.

The separation method using the separation apparatus pertaining to the present embodiment is the same as the procedure described in embodiment 1, differing from embodiment 1 only in that: after the surplus NaOH is washed away in the step (4), two ends of the filter column 21 are exchanged, the collection liquid is added, the filter column is kept still for more than 1min, the elution step can be carried out without blowing, the rotating speed of a centrifugal machine does not exceed 10000rpm during elution, and the filter column is centrifuged for at least 2 min. After collection, the DNA concentration can be detected by nanodrop, and pyrosequencing can be performed.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 95%.

Example 5

The only difference between this embodiment and embodiment 4 is that the filter membrane 22 can be placed at any position perpendicular to the separation channel 211, the two filter columns 21 are hollow and asymmetric, the diameter of the filter membrane 22 is slightly larger than that of the separation channel 211, and the two sides of the filter membrane 22 are provided with a squeeze membrane mechanism (not shown in the figure) to fix the filter membrane 22 against displacement.

When designing the primer, an affinity linker label for biotin-avidin binding can be selected, and any linker that can be labeled with DNA can be used to fix on the membrane, which is not described herein.

If an affinity linker with avidin is used, a membrane system with affinity adsorption can be selected as a membrane system for separating DNA single strands. The membrane system with affinity adsorption effect includes a silicon membrane substrate membrane, a magnetic particle membrane, an anion exchange material membrane, a nitrocellulose membrane or a polyamide membrane, and modified and coated magnetic beads and/or a silica membrane, etc., which are not limited herein.

The double-stranded DNA separation in the present invention can also adopt the conventional collection methods in the prior art, such as DNA single-stranded separation kit, etc., and the structure and method for separating and collecting DNA single-stranded DNA are all included in the protection scope of the present invention, and are not described herein again.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 95%.

Example 6

This example differs from example 1 only in that: the end face of the sample adding needle 41 is a plane, the diameter is 1.5mm, the surface finish is Ra 3.2, in the sample adding method, the moving speed of the sample adding needle 41 when leaving the dNTP reagent liquid in the step a is 50cm/s, the moving speed of the sample adding needle 41 when leaving the sequencing reaction liquid in the step b is 0.4cm/s, and the sample adding needle 41 moves up and down in the sequencing reaction liquid for 5 times and then leaves the sequencing reaction liquid.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 96%.

Example 7

This example differs from example 1 only in that: the end face of the sample adding needle 41 is a cone, the diameter is 1.6mm, the taper is 60 degrees, and the surface finish is Ra 9.8, in the sample adding method, the moving speed of the sample adding needle 41 when leaving the dNTP reagent liquid in the step a is 5cm/s, the moving speed of the sample adding needle 41 when leaving the sequencing reaction liquid in the step b is 4.5cm/s, and the sample adding needle moves up and down in the sequencing reaction liquid for 12 times and then leaves the sequencing reaction liquid.

The sequencing spectrogram and other experimental result data (sequencing time, loading repetition precision and uniformity) obtained in the example are consistent with the data obtained in the example 1 within the theoretical error range, and the loading repetition precision is 95%.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention. Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims (10)

1.A DNA sequencing device based on pyrosequencing is characterized by comprising a sample area, a reaction area and a detection area; the sample area comprises a separation disc (2) arranged on a centrifuge, the separation disc (2) comprises at least one DNA separation area, the separation area comprises a hollow filter column (21) internally provided with a filter membrane (22), the filter membrane (22) is made of polyethylene microspheres, the pore space among the microspheres is 10 mu m, 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 (22); the reaction area comprises a sample adding frame (4), dNTP reagent tanks (7) and a reaction tank (3), a plurality of sample adding needles (41) are fixedly arranged on the sample adding frame (4), the sample adding frame (4) reciprocates between the dNTP reagent tanks (7) and the reaction tank (3) through a linear moving device, a rotating shaft (42) is arranged at the center of the sample adding frame (4), the rotating shaft (42) drives the sample adding frame (4) to rotate through a motor, a lifting device is arranged between the sample adding frame (4) and the rotating shaft (42), and the sample adding frame (4) is lifted along the rotating shaft (42) through the lifting device; the reagent tank (7) is provided with a plurality of slots (71), the reagent tank (7) is provided with a substrate supply part, and the substrate supply part is used for conveying four nucleic acid substrates of dATP, dCTP, dGTP and dTTP of dNTP to the slots (71) through a conveying pipeline (1); the reaction tank (3) is provided with a plurality of reaction sites (31), and the reaction sites (31) extend to a detection area; the detection area comprises a bioluminescence detector (5) and a rotating platform (51), the bioluminescence detector (5) is detachably arranged on the rotating platform (51), and the rotating platform (51) drives the bioluminescence detector (5) to rotate through a motor; the sample area, the reaction area and the detection area are arranged on the analysis table (1), a manipulator for operation is arranged on the side surface of the analysis table (1), and the linear moving device is fixed on the analysis table (1) through a mounting frame (11).

2. The DNA sequencing device based on pyrophosphate sequencing according to claim 1, wherein the upper end of the rotating shaft (42) is provided with a fixed seat (44), and a bearing is arranged between the fixed seat (44) and the rotating shaft (42).

3. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 2, wherein said linear moving means comprises a moving slide (45) fixed on a mounting frame (11) and a moving slide (43) fixed on a fixed base (44).

4. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 2, wherein said lifting means comprises a lifting slide (46) fixed to the rotating shaft (42) and a lifting slide (47) fixed to the sample holder (4).

5. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 1, wherein said separation disc (2) is provided with a plurality of collection tubes (23), said filter column (21) is mounted in the collection tubes (23), and said filter membrane (22) is located at an end of one end of the filter column (21) or at a non-end of the filter column (21).

6. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 5, wherein the collection tube (23) is provided with a detachable upper cover (231), and the upper cover (231) is connected with the collection tube (23) by a connecting band (232).

7. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 1, wherein said sample holder (4) is circular, said sample pins (41) are uniformly distributed along the circumferential direction, and said reaction sites (31) are uniformly distributed on said reaction well (3) along the circumference having the same diameter as that of said sample pins (41).

8. The DNA sequencing apparatus according to claim 7, wherein the reaction sites (31) are made of a transparent material, and the bioluminescence detector (5) is located within a circle surrounded by all the reaction sites (31).

9. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 8, wherein said bioluminescent detector (5) is a CCD camera, and said rotary platform (51) is provided with a slot (511) for placing the CCD camera.

10. The pyrophosphate sequencing-based DNA sequencing apparatus according to claim 1, wherein said reaction region further comprises a wash bath (8) and a dry bath (6) mounted on the analysis stage (1), said wash bath (8) and dry bath (6) being located between the dNTP reagent bath (7) and the reaction bath (3).

Images