HK1246337B - Apparatus and method of sequencing dna based on pyrosequencing - Google Patents

Description

DNA sequencing device and sequencing method based on pyrophosphate sequencing

Technical Field

The present invention relates to the technical field of DNA sequencing, in particular to a DNA sequencing device based on pyrophosphate sequencing, belonging to the field of gene detection.

Background Art

1. Overview of Pyrosequencing

Pyrosequencing, developed in 1987, is a sequencing technology based on the detection of pyrophosphate (PPi) released during DNA synthesis. The pyrosequencing reaction, catalyzed by a series of enzymes, produces visible light proportional to the number of deoxynucleoside triphosphates (dNTPs) polymerized. DNA sequences can be determined by detecting this visible light. There are two methods for performing pyrosequencing: liquid-phase pyrosequencing and solid-phase pyrosequencing.

Liquid-phase pyrosequencing is an enzyme cascade chemiluminescent reaction in the same reaction system catalyzed by four enzymes. Its principle is: after the primer and template DNA are annealed, the polymerization of each dNTP on the primer DNA is coupled with the release of fluorescent signal under the synergistic action of DNA polymerase, ATP sulfurylase, luciferase and apyrase. By detecting the release and intensity of fluorescence, the purpose of real-time DNA sequence determination is achieved (Ma Yongping et al., Pyrosequencing Technology and Its Application in Molecular Biology [J]. Foreign Medical Molecular Biology, 2003, 25(2):115-117).

The liquid-phase pyrosequencing reaction system consists of a reaction substrate, a single strand to be tested, a specific sequencing primer, and an enzyme. The reaction substrates are 5'-phosphosulfate (APS) and luciferin. The liquid-phase pyrosequencing reaction involves the alternating addition of four dNTPs to the reaction system, with only one dNTP participating in each round. If the added dNTP pairs with the next base in the DNA template, it is added to the 3' end of the sequencing primer by DNA polymerase, releasing a molecule of pyrophosphate (PPi). Under the action of ATP sulfurylase, the generated PPi combines with APS to form ATP. Under the action of luciferase, the generated ATP combines with luciferin to form oxyluciferin, simultaneously generating visible light. If the added dNTP matches the next n consecutive identical bases in the DNA template, the reaction equation indicates that the intensity of the released visible light should be n times that of a single base match. In other words, the light intensity released during the reaction is proportional to the number of base matches. If the added dNTP does not match the next base in the DNA template, the above reaction will not occur and no visible light will be released. The dNTP and ATP that do not participate in the reaction are degraded by the action of the nucleotide degrading enzyme Apyrase.

The visible light released in each round of reaction is converted by a weak light detection device and then processed into a digital signal. After processing by PC software, a specific detection peak can be obtained. The height of the peak should be proportional to the number of matching bases.

After the previous round of reaction is completed, another dNTP is added and the above reaction is repeated. Finally, the DNA sequence information to be tested can be read based on the obtained light intensity peak graph.

It is important to note that deoxyguanosine monophosphate (dAMP), a degradation product of dATP, is an inhibitor of luciferase. As the reaction proceeds, its concentration increases, hindering the continued chemiluminescence reaction of pyrosequencing. This is also the main reason why pyrosequencing sequencing has a short sequencing length (usually 20 to 30 bp) (Shendure J, et al. Advanced sequencing technologies: Methods and goals, Nat. Rev Genet., 2004, 5(5): 335-44).

Solid-phase pyrosequencing is a chemiluminescent reaction catalyzed by three enzymes. Unlike liquid-phase pyrosequencing, it does not involve the enzyme apyrase. The solid-phase pyrosequencing reaction proceeds as follows: a DNA template bound to a primer is fixed to a support and remains in place during the reaction. After the addition of a single dNTP, a reaction occurs under the synergistic action of DNA polymerase, ATP sulfurylase, and luciferase. Except for the absence of degradation, the reaction is identical to liquid-phase pyrosequencing. A washing step is performed before the addition of the next dNTP to completely remove any residues from the previous reaction, preventing the accumulation of inhibitory products.

Usually, when people refer to pyrosequencing, they are referring to liquid-phase pyrosequencing, because its four-enzyme reaction system allows pyrosequencing to be easily performed in a single tube.

Ronaghi et al. used dATPαS to replace dATP to improve the signal-to-noise ratio of pyrophosphate sequencing (Ronaghi M. et al. Real-time DNA sequencing using detection of PPi release; Anal. Biochem, 1996, 242(1):84-89). This is because dATPαS can be used more efficiently by DNA polymerase than dATP, making it more conducive to reading T-rich regions. dATPαS is a mixture of two isomers, Sp-dATPαS and Rp-dATPαS, and polymerase can only use Sp-dATPαS. To achieve optimal reaction efficiency, the optimal concentration of Sp-dATPαS must be maintained in the reaction system, while the concentration of Rp-dATPαS is also increased. After dATPαS is degraded by Apyrase, it still produces a luciferase inhibitor, so the addition of dATPαS does not improve sequence reading ability.

Gharizadeh et al. improved this approach by adding only pure Sp-dATPαS to the reaction, rather than the useless Rp-dATPαS. This improved the reaction efficiency and significantly reduced the concentration of inhibitory products, allowing luciferase to maintain activity for a longer period of time. This increased the sequencing length of pyrosequencing to 50 to 100 bp, and the increased sequencing length also enabled many new applications of pyrosequencing technology (Gharizadeh B. et al., Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-0'-(1-thiotriphosphate)Sp-isomer; Anal Binchem, 2002. 301: 82-90).

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

Compared with the Sanger dideoxy chain termination sequencing method, pyrophosphate sequencing is fast, accurate, economical, and can detect in real time. It does not require gel electrophoresis or any special form of labeling and staining of DNA samples, and has high reproducibility. It can achieve a high degree of parallelism and automation.

2. Application Progress of Pyrosequencing Technology

1. Application in single nucleotide polymorphism research

Single Nucleotide Polymorphisms (SNPs) are third-generation genetic markers that have emerged in recent years. They refer to the presence of two different bases at a specific nucleotide position in the genome, with the least frequent of these two bases occurring in a population at least 1%. SNPs are the most common genetic polymorphisms in biological genomes, providing a series of markers within or near any gene under study. It is precisely these genomic polymorphisms, or differences in genomic sequence, that form the genetic basis for the susceptibility of individuals and populations to disease, and their varying responses to drugs and environmental factors.

SNP research involves two main aspects: first, the construction of a SNP database, primarily to identify all or part of the SNPs in the genome of a specific species. Second, the functional investigation of SNPs. Discovering SNPs is only the first step in SNP research; the study of SNP function is the ultimate goal. Sanger sequencing has become the mainstream technology for large-scale, accurate, and rapid SNP discovery. Pyrosequencing, excelling at short sequence sequencing and verification, is an excellent choice for sequence verification and frequency analysis of SNPs already in the database. Using pyrosequencing for SNP research can save time and reduce costs.

Nordfors et al. used Taqman fluorescence quantitative analysis and pyrosequencing to perform SNP genotyping on up to 1022 samples, obtaining similar results. This control experiment demonstrated that pyrosequencing is an efficient and highly accurate method for high-throughput, large-sample SNP analysis. Wasson et al. used pyrosequencing to analyze SNP allele frequencies in DNA pools. Rickert et al. used pyrosequencing to genotype tetraploid potatoes. Of the 94 polymorphic loci tested, 76 alleles could be identified using pyrosequencing, achieving an efficiency of 81%. Jiang Siwen et al. used pyrosequencing to identify haplotypes of the porcine mitochondrial cytochrome b gene. Yuan Jianlin et al. used pyrosequencing to analyze HLA-DRB genotypes. The experiment demonstrated that accurate genotyping could be achieved by comparing pyrosequencing results with gene sequences in the HLA database, suggesting that this method could be applied to donor/recipient screening in clinical organ transplantation.

2. Application in rapid identification of pathogenic microorganisms

Jonasson et al. used pyrosequencing to detect the 16S rRNA gene of pathogens and rapidly identified antibiotic-resistant bacteria in clinical specimens. Monstein et al. successfully used pyrosequencing to detect the variable V1 and V3 regions of the 16S rRNA gene of Helicobacter pylori, demonstrating that the technique is suitable for rapid identification and typing of clinical pathogen specimens. Unnerstad et al. used this technique to type 106 strains of Listeria monocytogenes of different serotypes. Pyrosequencing allows for the rapid sequencing of a large number of samples in a short period of time, demonstrating its remarkable parallelism and high efficiency. Gharizadeh et al. used this technique to identify and type 67 human papillomavirus samples, demonstrating that the technique is also well-suited for large-scale identification, typing, and mutation studies of pathogens such as HPV. Cheng Shaohui et al. extracted viral RNA from Vero-6 cells infected with the human SARS virus and used pyrosequencing to sequence multiple base mutation sites and analyze mutation frequencies. By sequencing and analyzing multiple potential mutation sites, they confirmed that the virus was the Beijing epidemic strain.

3. Application in etiology research

Kittles et al. used pyrosequencing to analyze CYP17 gene polymorphisms in three different populations: Nigerians, European Americans, and African Americans. They investigated the relationship between CYP17 gene polymorphisms and prostate cancer, as well as the clinical manifestations, in African Americans. The results showed that African Americans with the CC CYP17 genotype had a higher risk of prostate cancer than those with the TT CYP17 genotype, demonstrating that the C CYP17 gene polymorphism in this population is closely associated with prostate cancer incidence, placing them at high risk. Numerous clues have suggested a significant link between the COMT gene, located on chromosome 22q11, and the onset of schizophrenia, but research efforts have so far failed to provide conclusive evidence. Shifman et al. developed an effective method. Using pyrosequencing, they analyzed relevant single nucleotide polymorphisms in a large sample of Ashkenazi Jewish individuals and confirmed a strong association between schizophrenia and the CMOT gene. This method is also applicable to genetic analysis studies of other diseases.

4. Application in forensic identification

Sanger sequencing for mitochondrial DNA (mtDNA) variation analysis cannot accurately quantify mtDNA mixtures containing contaminants or DNA from multiple individuals. However, Andreasson et al. proposed a novel quantification method for mtDNA mixture analysis based on pyrosequencing technology, which can easily and accurately detect major and minor mtDNA components from mixed samples of forensic evidence. Balitzki-Korte used pyrosequencing technology to sequence and analyze the mitochondrial 12S rRNA gene, detecting a 20-bp fragment within a 149-bp gene fragment. By referencing database sequences, they were able to fully determine the biological origin of the object, for example, determining whether a piece of skin tissue came from a missing person or an animal.

3. Pyrophosphate Analysis Device and Development

The application of pyrosequencing technology relies on the research and development of pyrophosphate analysis devices. Regardless of the type of pyrophosphate analysis device, its main structure should consist of two parts: a reactor and a weak light detection unit. The reactor provides a place for the reaction to proceed, while the weak light detection unit detects the visible light emitted by the reaction. In the research and application of pyrosequencing technology, the reactors designed and used can be mainly divided into three categories: microplate reactors, microfluidic chip reactors, and microarray chip reactors.

While commercial pyrosequencing instruments have been commercialized overseas, there are few reports on research related to these instruments in China, and no corresponding products have been developed. A representative example of this international product is the PSQ96 from Pyrosequencing AB, launched in 2001. This system can simultaneously sequence 96 or 384 independent DNA samples. For sequences up to 300 base pairs in length, the system typically takes 1 hour and 45 minutes, achieving 99% accuracy and reliability. It offers the advantages of high throughput, speed, and cost-effectiveness. The PSQ96 system has been widely used in basic medical research and clinical molecular diagnostics.

Another representative example of international instrument research is the GenomeSequencer 20 (GS20), launched in 2005 by 454 Life Sciences in the United States. This instrument embodies a more technologically advanced approach to miniaturization, utilizing MEMS technology to utilize microfiltration chambers as the reaction environment for pyrosequencing reactions. This allows for the integration of an astonishing millions of reaction arrays into a 7cm x 8cm area, enabling each reaction chamber to independently and simultaneously perform sequencing cascade reactions. The instrument's highly sensitive and high-resolution CCD captures the faint fluorescent signals generated by each reaction chamber, ultimately providing sequence information for each DNA sample. The GS20 achieves high-density sequencing in just 4.5 hours, using parallel computation to determine the sequence information for each sample. This reduces reagent consumption, lowers sequencing costs, and opens the door to large-scale genome sequencing.

At present, domestic instrument research has just started, and the road to localization is long. Faced with various problems, after weighing the various hardware conditions required for the sequencing system, it is found that the primary problem and challenge is the development of the micro-amount subsystem.

IV. The Importance of the Sample Loading System in Pyrosequencing

Pyrosequencing technology and its products provide an ideal technical operation platform for high-throughput, low-cost, timely, rapid, and intuitive single nucleotide polymorphism research and clinical testing. It is a powerful tool for gene sequence analysis in the post-genomic era. Pyrosequencing technology is being accepted and adopted by more and more researchers. With the rise of pyrosequencing technology applications and the development of commercial pyrosequencing instruments internationally, the application of pyrosequencing technology in my country is booming. However, at this stage, there are several constraints on the application and promotion of pyrosequencing technology in China: (1) Existing commercial pyrosequencing instruments such as PSQ96 and GS20 are expensive; (2) The waiting time for commercial pyrosequencing services is long and inconvenient; (3) Although some laboratories are currently conducting research on devices such as pyrosequencing chips, and some laboratories have homemade, simple-structured pyrosequencing test devices, there is no low-end, inexpensive, commercialized sequence detection instrument based on pyrosequencing technology in China, which is a key issue restricting the application and development of pyrosequencing technology.

Pyrosequencing systems operate in a micro-volume environment, typically with a reaction volume of only 50 μL. The required amounts of reaction substrates, DNA templates, and other reagents, such as deoxynucleotides, are extremely small. Furthermore, the size of a single injection directly impacts the sustainability of the cyclic reaction. Excessive injections rapidly increase the reaction volume, leading to a rapid decrease in template concentration. Due to the nonlinear increase in reaction delay caused by diffusion, the resulting weak fluorescence signal stretches along the time axis and decreases in intensity along the vertical axis, ultimately significantly shortening the sequenceable length of the nucleic acid. It is generally considered acceptable to impact experimental results if the increase in reaction volume due to subsequent injections is within 10%. For example, if a 20-bp DNA fragment is to be sequenced, the permissible single injection volume in a 50 μL reaction volume should not exceed 0.3 μL.

In addition to sample addition accuracy, the accuracy of the sample addition intervals is equally important. Only by adding the dNTPs required for a single cycle at equal intervals can the degradation of residual dNTPs be consistent each time, thus minimizing the impact on the next reaction. Equal time intervals also provide a baseline for the signal in each cycle, facilitating subsequent automated analysis to calculate the number of nucleotides incorporated based on fluorescence signal intensity.

While domestically produced sample handling equipment is relatively abundant, existing micro-volume pipetting devices in China have shortcomings. For example, Shanghai FuRi's sample handling platform, a large-scale automated sample pool processing device, can handle sample loading, oscillation, and cleaning of standard 96-well plates. However, due to limitations in nozzle processing, this system has a minimum micro-precision of only 1μL, which cannot meet the nanoliter-level requirements of pyrophosphate sequencing. Research has shown that due to limitations in domestic application and manufacturing capabilities, domestically produced sample handling devices are unable to meet the high requirements for sample loading volume and repeatability required by pyrophosphate sequencing.

Ge Jianhui et al. from Southeast University disclosed a liquid-phase pyrophosphate analysis device with a weak light detection module and a micro-dNTP sampling module as key modules. The device disclosed a pressure-controlled micro-dNTP sampling module that can simultaneously sample 96 dNTP solutions, with a minimum sample volume of 1.2 μL and a maximum error of 13%. However, the signal noise is relatively large and further improvement is still needed (Ge Jianhui et al., Development of a Gene Detection Device Based on Pyrophosphate Sequencing, Southeast University, Master's Thesis, 2006).

Wang Chunlin et al. disclosed a micro-injection system for pyrophosphate nucleic acid sequencers using a piezoelectric ceramic nozzle. Driven by a stepper motor, this system can alternately inject four dNTP reagents into a 96-well standard plate. The system achieves an injection repeatability greater than 95%, and the minimum single injection volume can reach 0.1 μL. The micro-injection systems used in the two aforementioned preferred pyrophosphate nucleic acid sequencers are relatively complex, with injection needles prone to clogging. The dNTPs are injected into the sequencing reaction solution via various injection methods without contact, resulting in insufficient mixing and incomplete reaction. The system also requires a high dNTP dosage and can easily lead to inaccurate data. Furthermore, the system is cumbersome to assemble and disassemble, resulting in high costs and unsuitable for use under specialized conditions.

Pyrosequencing is a new DNA sequence analysis technique developed in recent years. It uses pyrophosphate released after the binding of nucleotides to the template to trigger an enzyme cascade reaction, which causes fluorescent light to be emitted and detected. It is an ideal genetic analysis platform, capable of performing not only DNA sequence analysis but also sequence-based single nucleotide polymorphism (SNP) detection and allele frequency determination. This technology is currently widely used in various fields, including medicine and biology.

Pyrosequencing is an enzymatic cascade chemiluminescent reaction catalyzed by four enzymes: DNA polymerase, ATPsulfurylase, luciferase, and apyrase. The substrates are adenosine 5'phosphosulfate (APS) and luciferin. The reaction system also includes a single-stranded DNA to be sequenced and a sequencing primer. During each round of sequencing, a dNTP is added. If the dNTP is paired with the template, the polymerase incorporates it into the primer strand, releasing an equimolar number of pyrophosphate groups (PPi). Sulfurylase catalyzes the conversion of ATP from ATP and PPi to oxyluciferin, which drives the luciferase-mediated conversion of luciferin to oxyluciferin. This generates a visible light signal proportional to the amount of ATP. This signal is converted by the Pyrogram ^™ into a peak whose height is proportional to the number of nucleotides incorporated in the reaction. The nucleotide sequence of the template DNA is recorded in real time based on the type of dNTP added and the intensity of the fluorescence signal. During the experiment, α-sulfated adenosine triphosphate (dATPαS) was used instead of dATP to effectively be utilized by DNA polymerase and not be recognized by luciferin. Because SpdATPαS can reduce the concentration of dATPαS degradation products, recent developments in single-stranded DNA binding protein (SSBP) and purified SpisomerdATPaS have effectively addressed the issue of using dATPαS degradation products to inhibit the activity of diphosphatases, enabling sequencing lengths up to 10 base pairs, expanding the application of this technology in genetics.

In pyrophosphate sequencing, the DNA sequence is determined by detecting luminescence through the use of staged complementary chain synthesis reactions and chemiluminescent reactions. The reaction tank containing the pyrophosphate generated by the complementary chain synthesis and the remaining nucleic acid substrate for complementary chain synthesis is moved to another reaction tank for luminescent reaction. During the movement of the reaction liquid, the remaining nucleic acid substrate is decomposed through an area fixed with an enzyme that decomposes the remaining nucleic acid substrate, and the pyrophosphate is converted into ATP and introduced into the chemiluminescent reaction tank. However, the disadvantage of the existing technology is that a large amount of substrate and enzyme must be added for the reaction to ensure that the reaction can be carried out completely, and then the excess substrate needs to be reacted and cleaned. This not only increases the reaction process and increases the difficulty of cleaning and reaction in each step, but also causes serious waste of reagents such as substrates, a long reaction time, and a waste of manpower and material resources, which invisibly reduces the promotion and use potential of pyrophosphate sequencing in the market.

Currently, the instruments used for pyrosequencing are mostly manufactured and sold by a few manufacturers, with instruments and reagents sold as a complete set. This makes sequencing very expensive, and testing and maintenance processes require specialized technicians, resulting in long cycles and high costs. The large reaction volume required further increases the cost, resulting in unstable test results and low repeatability. Therefore, it is imperative to independently design and develop a DNA sequencer suitable for pyrosequencing.

5. Overview of DNA Single-Strand Separation Technology

DNA single-strand separation technology is one of the most common separation techniques in the biomedical field. It is suitable for DNA sequencing and probe equipment of various scales for different nucleic acid samples and is widely used in biology, medicine, preventive medicine, zoology and botany, agriculture and animal husbandry, food and health, energy and chemical industry, environmental monitoring, and medical diagnosis and testing. In addition, DNA single-strand adsorption, extraction, and separation technology is widely used in the analysis, detection, separation, and purification of water quality, water sources, biomaterials, biological fluids (such as blood, serum, plasma, cerebrospinal fluid, urine, tears, sweat, digestive fluids, semen, secretions, tissue fluids, vomitus, and feces), tissue/cell and microbial lysates, proteins from different sources, nucleic acids, and other biological and chemical molecules, as well as drugs, and in the synthesis of oligonucleotides, peptides, lead compounds, and drugs. These technologies are closely related to people's daily lives and play a pivotal role in the biomedical field.

The common methods for separating single-stranded DNA in the biomedical field have the following shortcomings:

1. Heat denaturation or alkaline treatment. This method involves heating or alkali-treating the double-stranded PCR product. High temperatures or alkaline conditions can break hydrogen bonds between double-stranded DNA, resulting in single-stranded DNA. While this method is feasible and simple to perform, its low separation efficiency and purity have led to its gradual decline in use for single-stranded DNA purification, primarily for double-stranded DNA separation.

2. T7 reverse transcription. This method involves adding a T7 promoter to the 5' end of a PCR primer and using the purified PCR amplification product as a template to synthesize single-stranded RNA in vitro using T7 RNA polymerase for reverse transcription (Hughes, et al., Nat. Biotechnol., 2001, 19:342-347). While this method is feasible in principle, with a high DNA single-strand isolation rate and high purity, the entire isolation process requires two steps, which is inconvenient and time-consuming. Furthermore, strict control of RNase contamination is required, resulting in certain limitations.

3. Exonuclease method (Higuchi and Ochman, Nucleic Acids Res., 1989, 17:5865). Because one PCR primer is phosphorylated, the phosphorylated primer strand is not cleaved during exonuclease digestion of the PCR product. The enzyme is then heat-inactivated after digestion. This method also requires purification of the PCR product, a lengthy and inconvenient procedure. Furthermore, the yield of single-stranded DNA depends on the activity of the exonuclease, which is subject to significant uncontrollable factors and results are not stable. Consequently, this method has not been widely adopted and is not very versatile.

4. Denaturing high-performance liquid chromatography (DHPLC). Under partial denaturing conditions, DNA mutations are detected by the difference in retention time between heterozygous and homozygous diploids in the column. Heterologous DNA duplexes have different melting characteristics than homologous DNA duplexes. Under partial denaturing conditions, heterologous duplexes are more easily denatured due to the presence of mismatches, and their retention time in the chromatographic column is shorter than that of homologous duplexes. Therefore, they are eluted first, appearing as a double-peak or multi-peak elution curve in the chromatogram. Because one PCR primer is biotinylated, its PCR amplified strand is separated from the other normal strand during DHPLC (Dickman and Hornby, Anal. Biochem., 2000, 284:164-167). This method can directly obtain the desired single-stranded DNA from the double-stranded PCR product within 15 minutes, but its implementation requires very expensive equipment, making it difficult to popularize.

5. Magnetic bead capture method. Using nanotechnology to improve and modify the surface of superparamagnetic nanoparticles, superparamagnetic silica nanoparticles are prepared. These magnetic beads can specifically recognize and efficiently bind to nucleic acid molecules at a microscopic interface. Leveraging the superparamagnetic properties of silica nanoparticles, DNA and RNA can be separated from samples such as blood, animal tissue, food, and pathogenic microorganisms under the influence of chaotropic salts (such as guanidine hydrochloride and guanidine isothiocyanate) and an external magnetic field. The target single strands are then treated with sodium hydroxide to obtain the target single strands. This method is simple to use and time-efficient. The entire four-step extraction process can typically be completed within 36-40 minutes. It is safe and non-toxic, eliminating the use of toxic reagents such as benzene and chloroform used in traditional methods, minimizing harm to the operator and aligning with modern environmental protection concepts. The specific binding of the magnetic beads to the DNA single strands results in high purity and concentration of the extracted DNA single strands. However, the coated magnetic beads used in this method are relatively expensive, and separation requires a magnetic stand, which is not only costly but also inconvenient, thus limiting the widespread adoption of this technology.

6. Asymmetric PCR. All of the above methods require additional processing after PCR, while asymmetric PCR can prepare single-stranded DNA at the same time as PCR amplification. Conventional asymmetric PCR uses two unequal amounts of primers and performs normal amplification in the initial cycle. As the number of cycles increases, the primer in small amounts is gradually exhausted, while the excess primer can continue to linearly amplify to generate single-stranded DNA (Gyllensten and Erlich, Proc. Natl. Acad. Sci. U.S.A., 1988, 85: 7652-7656). This method has high hybridization sensitivity and specificity, and is easier to operate, but the ratio of its primers needs to be optimized, and the chance of non-specific amplification increases. In addition, the DNA single-strand separation process requires electrophoresis, the separation procedure is complicated, and diffuse bands are often seen during electrophoresis. Its time-consuming and inconvenient nature is obvious.

The above-mentioned several separation methods all have certain limitations. Therefore, in order to meet the requirements of operability and economy for separating single-stranded DNA, the existing single-stranded DNA extraction methods use an integrated extraction workstation. An affinity linker with streptavidin is bound to the double-stranded DNA. The workstation has a filtration needle and a matching pump. The bound DNA affinity linker is adsorbed on the lower part of the filter membrane in the filtration needle through filtration. The workstation is equipped with a track and related systems. After filtration, the filtration needle is moved to a tray filled with NaOH. The double helix is treated with alkali to obtain the single-stranded DNA. After filtration, it is cleaned and collected again. The filtration needles are generally arranged in groups of 24 (4*6). When used, sufficient sample or reagent must be ensured to ensure the normal operation of the workstation. Therefore, this method of collecting single-stranded DNA is very inflexible. It can only be added to the workstation in fixed amounts. A large amount of loss occurs during the multiple filtration and transfer processes, which is very unfavorable for micro-collection. In addition, the filtration needle group needs to work simultaneously, which places certain volume requirements on each component of the workstation, and the entire workstation takes up a lot of space. The huge system requires repeated pipetting back and forth on the micro-separation column during the DNA single-strand separation operation, which is very cumbersome to operate. Not only is the separation cycle long and the efficiency low, but the overall equipment is expensive, resulting in high costs for DNA single-strand separation. It also requires a large amount of reagents and other resources, which is extremely uneconomical. In addition, the filtration needles in this workstation are made of metal, which is expensive and is often reused after processing. Therefore, it is easy to cause cross-contamination between residues, which is not reliable and will cause certain interference and impact on the accuracy of separation and detection results. In addition, during the solution extraction process, some residual solution will adhere to the wall, so that a certain amount of target DNA single strands cannot be adsorbed by the micro-separation column, resulting in a decrease in the proportion of DNA single strands obtained, affecting the separation rate and causing waste. Therefore, the problem of high-quality and efficient DNA single-strand separation for pyrophosphate sequencing needs to be solved urgently.

Summary of the Invention

In view of the above problems existing in the prior art, the object of the present invention is to provide a DNA sequencing device based on pyrophosphate sequencing that is easy to operate and can perform rapid detection.

In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

A DNA sequencing device based on pyrosequencing, comprising a sample area, a reaction area, and a detection area;

The sample area includes a rotatable separation disk, which includes at least one DNA separation area, and the separation area includes a hollow filter column with a filter membrane inside. The DNA single strand connected to the affinity linker and the DNA single strand not connected to the affinity linker are separated after filtration through the filter membrane;

The reaction area includes a sample loading rack, a dNTP reagent tank, and a reaction tank. A plurality of sample loading needles are fixed on the sample loading rack. The sample loading rack reciprocates between the dNTP reagent tank and the reaction tank via a linear motion device. A rotating shaft is provided at the center of the sample loading rack, and the rotating shaft drives the sample loading rack to rotate via a motor. A lifting device is provided between the sample loading rack and the rotating shaft, and the sample loading rack is lifted and lowered along the rotating shaft via the lifting device. The reaction tank is provided with a plurality of reaction positions, and the reaction positions extend to the detection area.

The detection area includes a bioluminescence detector and a rotating table. The bioluminescence detector is detachably mounted on the rotating table, and the rotating table drives the bioluminescence detector to rotate via a motor. The sample area, reaction area, and detection area are mounted on an analysis table. A manipulator for operation is provided on the side of the analysis table. The linear motion device is fixed to the analysis table via a mounting bracket.

As a further improvement of the above technical solution:

Preferably, a fixing seat is provided at the upper end of the rotating shaft, and a bearing is provided between the fixing seat and the rotating shaft.

Furthermore, the linear moving device includes a movable slide rail fixed on the mounting frame and a movable slider fixed on the fixing seat.

Furthermore, the lifting device includes a lifting rail fixed to the rotating shaft and a lifting slider fixed to the sample loading rack.

Preferably, the filter membrane is arranged at one end of the filter column. In other words, the filter membrane constitutes the bottom end of the filter column, and the filter surface of the DNA separation zone is arranged at the bottom of the separation column, that is, the separated single-stranded DNA is on the filter membrane.

Furthermore, as a preferred embodiment, the outer diameter of the filter column is equal to the inner diameter of the collection tube, and the filter column and the collection tube can be kept in a relatively stable state through friction, without the need for an external structure to limit and fix the filter column.

In another preferred embodiment, the separation disc is provided with a plurality of collecting tubes, the filter column is installed in the collecting tube, and the filter membrane is located at a non-end portion of the filter column.

Furthermore, the filter membrane is a polymer nanosphere structure, and the pore size between the polymer nanospheres is smaller than the diameter of the affinity linker.

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

After separation, the single-stranded DNA on the filter membrane is a single strand connected to the affinity linker. This strand can be eluted from the filter membrane if needed. The filtrate contains the complementary strand without the affinity linker. The single-stranded DNA in the filtrate can be collected for reverse sequencing. When the strand with the affinity linker on the filter membrane needs to be collected, the collection tube can be a recycling tube. In this case, the collection tube only serves to recover wastewater, reducing costs and environmentally friendly waste.

The sample loading rack is circular, the sample loading needles are evenly distributed along the circumference, and the reaction positions are evenly distributed on the reaction tank along the circumference of a circle having the same diameter as the sample loading needles.

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

The bioluminescence detector is a CCD camera, and the rotating platform is provided with a slot for placing the CCD camera.

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

In response to the above-mentioned problems in the prior art, the advantages of the DNA sequencing device based on pyrophosphate sequencing of the present invention are:

(1) DNA single strands are extracted by filtering and centrifuging, achieving economical and miniaturized collection of one sample at a time, and providing a high-efficiency and low-loss rapid DNA single strand separation method and device. This method is simple and easy to operate, with a short time to obtain the target sample and high efficiency. It can be used to collect and extract trace DNA single strands, and the sample loss is almost negligible. It uses less reagents and has low requirements for equipment. No water pump is required during the separation process, which greatly simplifies the equipment configuration, reduces the total equipment cost, effectively simplifies the operation steps, shortens the operation time, reduces the work intensity, and improves the work efficiency. The use of a filter column that matches a conventional centrifuge tube not only ensures the quality and efficiency of the separation, but also makes the separation process simple and easy to implement. The filter membrane of the filter column separates the DNA single strands by physical means, reducing the difficulty and condition requirements of the separation. The filter membrane is composed of homogeneous materials with the same structure and can be used interchangeably in both directions, which increases the design possibilities of the filter column. It can not only be made into a hollow structure with a filter membrane at one end, enriching the structure and type of the DNA single-strand separation device, but also increasing the application occasions of the DNA single-strand separation device of the present invention while ensuring the same function, avoiding the single product structure and significantly enhancing the adaptability; and it can adopt an upper and lower symmetrical structure and can be interchanged and used in conjunction. The present DNA single-strand separation device adopts inexpensive PC plastic, which is economical and practical. The funnel-shaped inner cavity design is conducive to the aggregation and guidance of the reaction liquid, making the reaction liquid separation more sufficient and complete, obtaining a higher proportion of the target DNA single strand, ensuring a higher separation rate, and avoiding waste. In addition, the present DNA single-strand separation device is also suitable for commercially available centrifuge tubes. The standardized design makes the present DNA single-strand separation device extremely versatile and widely applicable, so its application prospects are very broad.

(2) The present invention uses a simple-structured sample needle to achieve micro-sampling of dNTP reagents, abandoning the transmission hollow needle tube suction-spraying sample delivery method. Micro-sampling and loading can be achieved only by controlling the adsorption force between the sample needle and the liquid and the difference in its movement speed in different liquids. Moreover, stirring can be achieved by moving the sample needle up and down several times in the sequencing reaction liquid, making the enzyme reaction more complete and the result more accurate. The sample delivery method and its device are suitable for any detection device, are easy to disassemble, simple and convenient to clean, and will not cause the problems of needle clogging and other shortcomings of the sample delivery devices in the prior art. The sample delivery repeatability is greater than 95%, and the minimum sample delivery volume can reach 0.1 μL. The sample delivery uniformity is good, the sequencing time is greatly shortened, and the result accuracy is extremely high. In addition, the sample nucleic acid sequence can be qualitatively and quantitatively detected by the detection device used in conjunction with it. Therefore, its application prospects are very broad.

(3) The DNA sequencing device based on pyrophosphate sequencing of the present invention uses a robot to control the entire process, is infection-free, has high accuracy, and improves efficiency and precision.

In summary, the DNA sequencing device based on pyrophosphate sequencing provided by the present invention reduces the amount of substrate and enzyme system used, has accurate detection, fast reaction speed, high integration, and can simultaneously detect one or more SNP sites and single-stranded DNA fragments. The enzyme system and substrate have broken the monopoly of some manufacturers, and the price has been greatly reduced. The analysis device has a precise structure and requires low amounts of DNA fragments and substrates to be tested, which reduces the detection cost of pyrophosphate sequencing and expands the scope of application of pyrophosphate sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic structural diagram of a DNA sequencing device based on pyrophosphate sequencing according to the present invention (the separation disk is not shown).

FIG2 is a schematic diagram of the top structure of FIG1 .

FIG3 is a preferred embodiment of the use of the DNA separation region for pyrophosphate provided by the present invention.

FIG4 is a schematic structural diagram of the filter column in Example 1 of the present invention.

FIG5 is a schematic structural diagram of the filter column in Example 2 of the present invention.

FIG6 is a schematic structural diagram of the filter column in Example 3 of the present invention.

FIG7 is a schematic structural diagram of the filter column in Example 4 of the present invention.

FIG8 is a schematic diagram of a sample loading needle in a sample loading device structure for a pyrophosphate sequencing system provided by the present invention.

Description of the numbers in the figure:

1. Analysis table; 11. Mounting frame; 2. Separation plate; 21. Filter column; 211. Separation channel; 22. Filter membrane; 23. Collection tube; 231. Upper cover; 232. Connecting belt; 3. Reaction tank; 31. Reaction position; 4. Sample loading rack; 41. Sample loading needle; 42. Rotating shaft; 43. Moving slider; 44. Fixed seat; 45. Moving slide; 46. Lifting slide; 47. Lifting slider; 5. Bioluminescence detector; 51. Rotating table; 511. Card slot; 6. Drying tank; 7. dNTP reagent tank; 71. Slot position; 8. Cleaning tank.

DETAILED DESCRIPTION

The present invention will be further described in detail and completely below with reference to the embodiments.

Example 1

Figures 1 to 4 illustrate a first embodiment of a DNA sequencing device based on pyrophosphate sequencing according to the present invention. The DNA sequencing device based on pyrophosphate sequencing according to the present invention can be used for pyrophosphate sequencing to detect and analyze DNA sequences. The DNA sequence to be sequenced is the target sequence, and the target DNA sequence is amplified and then subjected to pyrophosphate sequencing. The DNA sequencing device of this embodiment includes a sample area, a reaction area, and a detection area. The sample area, reaction area, and detection area are mounted on an analysis platform 1, and the sample area, reaction area, and detection area are monitored and controlled by a control area. The entire analysis process is operated by a robotic arm.

Before pyrosequencing, the DNA template to be sequenced must be amplified to achieve the required DNA concentration. When designing amplification primers, they are equipped with an affinity linker, preferably biotin, and are preferably labeled at one end of the target DNA. PCR amplification can be performed using existing techniques.

The sample area includes a separation tray 2, which is placed in a centrifuge. After target DNA amplification, the target DNA is double-stranded. Pyrophosphate sequencing requires single-stranded DNA. The separation tray 2 includes at least one DNA separation zone, which utilizes physical filtration. This zone includes a hollow filter column 21 with a filter membrane 22 inside. The filter membrane 22 is a polymer nanosphere structure. Because the pore size between the polymer nanospheres is smaller than the diameter of the affinity linker, the DNA single strand labeled with the affinity linker is retained on the membrane. The labeled DNA single strand or its complementary strand is then collected for pyrophosphate sequencing as needed for sequencing.

In this embodiment, the separation disc 2 is provided with a plurality of collection tubes 23, the filter column 21 is installed in the collection tube 23, and the filter membrane 22 is located at 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 tube 23.

After alkaline hydrolysis, the double-stranded DNA leaves one chain of the affinity linker on the filter membrane 22. To collect this chain, simply add a collection liquid into the filter column 21 and collect it. If the complementary chain needs to be collected, the filtrate can be collected and the complementary chain in the filtrate can be collected.

As shown in Figure 3, the collection tube 23 is used to collect waste liquid generated during centrifugation. After each step, it needs to be promptly dumped to prevent cross contamination. The upper end of the collection tube 23 is cylindrical, the lower end is conical, and the bottom is spherical. As another preferred embodiment, the collection tube 23 is equipped with a top cover 231, which is detachably connected to the collection tube 23 via a connecting band 232. Due to the connection of the connecting band 232, after the filter column 21 is installed, the top cover 231 can also be tightly fastened to the filter column 21 or the collection tube 23. The function of the top cover 231 is to prevent the liquid from splashing out of the filter column 21 during centrifugation, causing loss and contamination.

The preferred material of the filter membrane 22 in this embodiment is polyethylene microspheres, and the pores between the microspheres are preferably 10 μm, which is smaller than the diameter of the affinity linker. The single chain with the affinity linker can be directly retained on the membrane through physical filtration, and the chain not connected to the affinity linker can be filtered out. It has good adsorption and elution effects, high DNA recovery rate, and low raw material price and is environmentally friendly.

As shown in FIG4 , to prevent the filter membrane 22 from moving during pipetting or centrifugation, a membrane pressing device is preferably provided above the filter membrane 22. The membrane pressing device includes a spacer and/or a membrane pressing frame. The liquid to be separated and purified first passes through the spacer before coming into contact with the filter membrane 22. The spacer is preferably made of a fibrous material that is resistant to acids, bases, and most organic solvents and does not adsorb most biomolecules.

Preferably, a film pressing frame is provided above the gasket on the side that does not contact the filter membrane 22. The film pressing frame is made of the same material as the filter column 21 and compresses the filter membrane 22 through mechanical pressure so that the filter membrane 22 will not move during use such as blowing or centrifugation, causing collection loss.

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

The reaction area includes a sample loading rack 4 , a dNTP reagent tank 7 , a cleaning tank 8 , a drying tank 6 and a reaction tank 3 . The cleaning tank 8 and the drying tank 6 are located between the dNTP reagent tank 7 and the reaction tank 3 .

The dNTP reagent tank 7 is provided with four slots 71. Before the reaction, the substrate supply unit transports different nucleic acid substrates of dNTP to the slots 71 respectively through a pipeline. The substrate supply unit is used to provide dNTPs for hybridization reaction with the target DNA, providing an environmental basis for the hybridization reaction. The dNTPs used for pyrophosphate sequencing include four nucleic acid substrates: dATP, dCTP, dGTP, and dTTP. The substrates are used for hybridization with the target DNA, and it is necessary to add a related enzyme system catalyzed reaction in the reaction system, specifically DNA polymerase. When performing the complementary chain synthesis reaction, the byproduct pyrophosphate obtained during the complementary chain synthesis is converted into ATP. In the presence of luciferase, ATP and luciferin are reacted to emit light. If complementary chain synthesis occurs, pyrophosphate is produced, and the result is luminous. Therefore, by monitoring it, it can be learned that the complementary chain synthesis occurs, that is, the base types incorporated, and thus the DNA sequence is determined.

A plurality of sample loading needles 41 are fixedly mounted on the sample loading rack 4. The sample loading rack 4 is circular, and the sample loading needles 41 are evenly distributed along the circumference. The sample loading rack 4 reciprocates between the dNTP reagent tank 7, the cleaning tank 8, the drying tank 6, and the reaction tank 3 via a linear motion device, which is fixed to the analysis platform 1 via a mounting bracket 11. A rotating shaft 42 is provided at the center of the sample loading rack 4. The rotating shaft 42 is driven by a motor to rotate the sample loading rack 4. A lifting device is provided between the sample loading rack 4 and the rotating shaft 42, and the sample loading rack 4 is lifted and lowered along the rotating shaft 42 by the lifting device. The sample loading needles 41 are solid needles used to transfer dNTP reagents. The sample loading needles 41 are fixed in the through-holes of the sample loading rack 4. The single-stranded DNA is transferred to the reaction site 31 according to the required amount of the reaction system. After the enzyme system is added, dNTPs are added in sequence. The order of addition is not limited. For each site, each of the four substrates is added once. A fixed seat 44 is located at the upper end of the rotating shaft 42. A bearing is provided between the fixed seat 44 and the rotating shaft 42. When the rotating shaft 42 rotates, the fixed seat 44 does not rotate with it. The linear motion device includes a movable slide 45 fixed to the mounting frame 11 and movable sliders 43 fixed to both sides of the fixed seat 44. The lifting device includes a lifting slide 46 fixed to the rotating shaft 42 and a lifting slider 47 fixed to the sample loading rack 4. The linear motion device and the lifting device utilize linear guide rails.

The reaction tank 3 is provided with a plurality of reaction sites 31 , which are made of a transparent material and extend to the detection area. The reaction sites 31 are evenly distributed on the reaction tank 3 along a circle having the same diameter as the sample injection needle 41 .

The detection area includes a bioluminescence detector 5 and a rotating stage 51. The bioluminescence detector 5 is a CCD camera connected to a computer and displays the detected spectra. The rotating stage 51 is provided with a slot 511 for the CCD camera. The CCD camera is located within the circle formed by all reaction sites 31. The rotating stage 51 is driven by a motor to rotate the bioluminescence detector 5.

The configuration of the DNA sequencing system based on pyrophosphate sequencing of the present invention can better transfer substrate and DNA, and reduce loss during the transfer process.

The shape of the reaction area is not limited, and the specific structure within the analysis device can be designed and adjusted according to the needs of the detection. Any structural shape and relative position that can achieve pyrophosphate sequencing should be considered to fall within the scope of protection of the present invention.

Add the substrate to the reaction zone. At this point, the DNA single strand to be tested and other required reaction system reagents have been added to the reaction zone. Based on the amount of 5ul required for detection, the reaction system is as follows:

The enzyme blend includes:

The substrate mix includes:

APS 0.4mg/L;

Firefly luciferin 0.4mmol/L.

During the reaction process, the optimal pH for enzyme activity is selected at each step. After the reaction, the pH within the reaction system needs to be adjusted to accommodate other reactions. The reaction conditions for the specific reaction system can be determined by one of ordinary skill in the art based on existing technology. For example, when apyrase is included in the wash step to remove excess nucleotide species and ATP, due to the continuity of the treatment steps, a buffer can be used with the apyrase at a pH that optimizes the apyrase's cleanliness level. Then, in the next nucleotide incorporation step, a polymerase can be used with a different buffer having the optimal pH conditions for the polymerase to optimize polymerase cleanliness. In addition, each optimal buffer can include preferred counterions for each enzyme, such as Ca2 ⁺ for the apyrase buffer and Mg2 ⁺ for the polymerase buffer.

The sequencing results are determined by bioluminescence, and any other device capable of detecting bioluminescence can be used as a detection device without limitation.

The pyrophosphate sequencing-based DNA sequencing device in the embodiment of the present invention also includes liquid supply and discharge channels between the sample storage devices. The liquid supply channel is used to transport reagents and samples to be reacted, and the liquid discharge channel is used to discharge the liquid after the reaction is completed or after centrifugation to the collection component.

In the analysis device of the present invention, it is necessary to ensure that all operations are carried out in the most suitable environment during the reaction, and the enzyme system needs to react in the reaction system with the highest activity to ensure the complete reaction. Therefore, the control area is provided with several components that can ensure that these reactions can be carried out efficiently and orderly, including centrifuges, pH meters, heating components, control signal transmission components and other regular structures of sequencing equipment.

The systems and methods of embodiments of the present invention may include performing some design, analysis, or other operations 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 by sequencing result systems and methods, wherein the processing and analysis embodiments are executed 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. Computers typically include known components, such as a processor, an operating system, system memory, a memory storage device, input/output controllers, input/output devices, and a display. Those skilled in the relevant art will appreciate that there are many possible computer configurations and components, and that high-speed memory, data sharing units, and many other devices may also be included.

A display may include a display that provides visual information, such information typically organized logically and/or physically into 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 Interface" (commonly referred to as a GUI), which may provide one or more graphical displays to a user. The interface is typically capable of accepting user input using selection or input methods known to those skilled in the relevant art.

In the same or alternative embodiments, an application on a computer may utilize an interface including a so-called "command line interface" (commonly referred to as a CLI). A CLI typically provides a text-based interface for interaction between the application and the user. Typically, a command line interface displays output and receives input in the form of lines of text via a display. For example, certain execution processes 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 using an object-oriented programming architecture, such as the Microsoft .NET Framework.

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

The processor usually executes an operating system, and the operating system can be any operating system in the prior art. As long as it can be used by those skilled in the art to process test results and data, it is considered to be included in the scope of protection.

The DNA sequencing device based on pyrosequencing in the present invention can be widely used for the determination, diagnosis, and inspection of DNA sequences, the determination and diagnosis of SNP sites, etc., and can realize the simultaneous sequencing of a certain number of samples. The principle has low requirements for sequencing conditions and does not require the addition of expensive experimental reagents such as excitation light sources and fluorescent agents. It can realize simple and inexpensive DNA sequencing work, and the detection results are stable and the accuracy is high. It overcomes the shortcomings of previous equipment that cannot fully proceed or the complementary chain synthesis reaction proceeds excessively. The reaction volume is small and the reaction stage can be completed in a shorter time.

The analysis method using the DNA sequencing device and system based on pyrosequencing provided by the present invention includes the following steps, wherein the undisclosed parts can refer to the prior art:

(1) Binding: The amplified DNA fragments are bound to agarose beads. The length of the DNA fragments is 10 to 20 kb. The minimum amount of DNA loaded should be no less than 500 ng. The diameter of the agarose beads is 30 μm, and the surface is coated with biotin and streptavidin. The amplified DNA fragments spontaneously and specifically bind to the streptavidin-coated agarose beads.

(2) Centrifugation: The combined double-stranded DNA is drawn into the filter column 21. The filter column 21 is pre-loaded into the collection tube 23 and placed in a centrifuge for 1 minute at 12,000 rpm to remove excess solvent. The collection tube 23 is a 1.5 mL centrifuge tube. The filter column 21 is a cylinder with an open upper end and a semi-closed lower end. A boss is provided on the outer edge of the upper opening of the filter column 21 to fix it on the collection tube 23. A filter membrane 22 is built into the lower end of the filter column 21. The diameter of the filter column 21 is 4.5 mm, and the diameter of the filter membrane 22 is 4.7 mm. The filter membrane 22 is forcibly pressed into the filter column 21 to make it fit tightly without leaving any gaps.

(3) Washing: Add an appropriate amount of 70-80% ethanol to wash away the residual Taq enzyme and other amplification reagents, gently pipette to mix, and then centrifuge at 12000 rpm for 1 min.

(4) Alkaline hydrolysis: Add 0.4 M NaOH and 1 M NaCl to unwind the double-stranded helix, gently pipette to mix, and then centrifuge at 12,000 rpm for 1 min.

(5) Adjust the pH value: Add an appropriate amount of elution buffer or ultrapure water to wash away residual NaOH, balance the pH value to neutral, gently pipette to mix, and centrifuge at 12000 rpm for 1 min.

(6) Collection: Add a collection solution such as ultrapure water, and gently blow until the DNA single strand is completely suspended. After aspirating, use it for pyrophosphate sequencing. Add the aspirated DNA single strand to a reaction site 31 and add the enzyme required for the reaction.

(7) Sample loading: Move the sample loading rack 4 to above the dNTP reagent tank 7, lower the sample loading rack 4, and dip any dNTP reagent through the sample loading needle 41 so that the dNTP reagent is attached to the periphery of the sample loading needle 41.

(8) Reaction: Move the sample loading rack 4 to the top of the reaction tank 3, rotate the sample loading rack 4 so that the sample loading needle 41 dipped in the dNTP reagent is aligned with the reaction position 31 to be tested, lower the sample loading rack 4, insert the sample loading needle 41 attached with the dNTP reagent into the sequencing reaction solution, and then remove the sample loading needle 41 from the sequencing reaction solution.

To complete sequencing, the order in which the four dNTP reagents are added to the sequencing reaction solution is preferably determined according to any or multiple sequences to be tested. For example, when only detecting whether a single base is mutated, the position of the base to be tested can be determined based on the known preceding and following sequences, and then the above four addition processes (adding the four dNTP reagents) can be repeated sequentially, and the bases can be detected by adding them to the same sequencing reaction solution.

When the sample loading needle 41 leaves the sequencing reaction solution, the sample loading rack 4 moves to the cleaning tank 8 to clean the sample loading needle 41 , and then dries the cleaned sample loading needle 41 before dipping it into the next dNTP reagent.

(9) Conclusion: The CCD camera is connected to a computer to take pictures and display the detected spectrum.

The solutions, parameters, and other separation conditions involved in the DNA separation process in this example are all preferred embodiments of this example. Any experimental conditions, parameters, and solutions that can play a corresponding role with reference to the prior art can be used in the separation and purification process involved in the present invention. The specific parameters and solution selections in this example should not be construed as limiting the present invention.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 96%.

Example 2

Since a chain collected on the filter membrane 22 needs to be sucked out or poured out, this collection method may cause a certain amount of collection loss. Therefore, the inventors preferably set the filter column 21 to a double-headed structure with interchangeable ends. The filter membrane 22 is arranged on the non-end surface of the filter column 21. When collection is required, the two ends of the filter column 21 are interchanged and conventional methods such as centrifugation are used to elute all the DNA single chains on the filter membrane 22, thereby reducing sample loss.

As shown in FIG5 , 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 symmetrical structure, and the filter membrane 22 is vertically located in the middle of the hollow column. The filter column 21 can be used interchangeably at both ends. The inner diameter of the hollow column is 4.5 mm, and the diameter of the filter membrane 22 is 4.7 mm. The filter membrane 22 is forcibly pressed into the filter column 21 to fit tightly without leaving any gaps. A filter membrane 22 limiting and pressing mechanism (not shown) is added to the column to fix the filter membrane 22 and prevent it from moving. Since the DNA single-strand separation device is identical at both ends in terms of structure and function, there is no need to distinguish between the liquid inlet and liquid outlet ends. During use, either end can be used.

After washing away excess NaOH in step (4), swap the ends of the filter column 21, add the collection solution, and allow it to rest for at least 1 minute. Elution can then be performed without pipetting. During elution, the centrifuge speed should not exceed 10,000 rpm, and the centrifugation should be performed for at least 2 minutes. After collection, the DNA concentration can be measured using a nanodrop assay and pyrophosphate sequencing can be performed.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 95%.

Example 3

As shown in Figure 6 , this embodiment differs from Example 1 only in that the filter columns 21 of the DNA single-strand separation device in Example 3 employ a spatially truncated cone-shaped structure. The filter membrane 22 is positioned on the common top surface of the two truncated cone-shaped filter columns 21. The diameter of the top surface of the truncated cone-shaped filter columns 21 matches the diameter of the filter membrane 22. A membrane-pressing mechanism (not shown) is provided on either side of the filter membrane 22 to secure the filter membrane 22 against displacement. A boss is provided on the sidewall of the open lower end of the truncated cone-shaped filter column to ensure that it can be secured to the boss of the collection tube 23 when the two ends are swapped.

The separation method using the separation device of this embodiment is the same as that described in Example 1, with the only difference from Example 1 being that, after washing away excess NaOH in step (4), the ends of the filter column 21 are swapped, the collection solution is added, and the column is allowed to rest for at least 1 minute. Elution can then be performed without pipetting. During elution, the centrifuge speed does not exceed 10,000 rpm, and the centrifugation is performed for at least 2 minutes. After collection, the DNA concentration can be measured using a nanodrop assay and pyrophosphate sequencing can be performed.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 95%.

Example 4

As shown in Figure 7, the only difference between this embodiment and Example 1 is that, in order to better gather and guide the reaction liquid, make the reaction liquid separation more sufficient and complete, and avoid filtration dead corners, this embodiment adds a separation channel 211 on the basis of Example 1. The separation channel 211 is arranged between the two filter columns 21 and is connected to the two. The two filter columns 21 and the separation channel 211 share a common central axis. The filter membrane 22 is arranged on the symmetrical plane of the separation channel 211. A membrane pressing mechanism (not shown in the figure) is provided on both sides of the filter membrane 22 to fix the filter membrane 22 so that it cannot be displaced. A boss is provided in the middle of the two filter columns 21 to ensure that the two ends can be fixed on the boss of the collection tube 23 when swapped.

The end of the filter column 21 connected to the separation channel 211 is in the shape of an inverted truncated cone, and together they form a funnel-shaped spatial structure. The bottom diameter of the inverted truncated cone is consistent with the inner diameter of the filter column 21, and the top diameter is consistent with the diameter of the separation channel 211. This structure facilitates the aggregation and guidance of the reaction solution, resulting in more complete and sufficient separation of the reaction solution. Therefore, the DNA single-strand separation device in this embodiment, compared with Example 2, achieves more thorough DNA single-strand filtration separation and significantly improves separation efficiency.

The separation method using the separation device of this embodiment is the same as that described in Example 1, with the only difference from Example 1 being that, after washing away excess NaOH in step (4), the ends of the filter column 21 are swapped, the collection solution is added, and the column is allowed to rest for at least 1 minute. Elution can then be performed without pipetting. During elution, the centrifuge speed does not exceed 10,000 rpm, and the centrifugation is performed for at least 2 minutes. After collection, the DNA concentration can be measured using a nanodrop assay and pyrophosphate sequencing can be performed.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 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 asymmetric structures, the diameter of the filter membrane 22 is slightly larger than the separation channel 211, and a membrane pressing mechanism (not shown in the figure) is provided on both sides of the filter membrane 22 to fix the filter membrane 22 so that it cannot be displaced.

When designing primers, you can also choose to use a biotin-avidin-bound affinity linker label. Any linker that can be used to label DNA can be used for fixation on the membrane and will not be described in detail here.

If an affinity linker with avidin is used, membranes with affinity adsorption can also be used for DNA single-strand separation. Membranes with affinity adsorption include, but are not limited to, silica matrix membranes, magnetic particle membranes, anion exchange membranes, nitrocellulose membranes, polyamide membranes, and modified or coated magnetic beads and/or silica membranes.

The double-stranded DNA separation in the present invention can also adopt conventional collection methods in the prior art, such as a DNA single-strand separation kit, etc. As long as the structure and method can be used to separate and collect single-stranded DNA, they should be included in the scope of protection of the present invention and will not be described in detail here.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 95%.

Example 6

The only difference between this embodiment and Example 1 is that the end face of the sample injection needle 41 is flat, has a diameter of 1.5 mm, and a surface finish of Ra 3.2. In addition, in step a of the sample injection method, the movement speed of the sample injection needle 41 when leaving the dNTP reagent solution is 50 cm/s, and in step b, the movement speed of the sample injection needle 41 when leaving the sequencing reaction solution is 0.4 cm/s. The sample injection needle 41 moves up and down in the sequencing reaction solution five times before leaving the sequencing reaction solution.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 96%.

Example 7

The only difference between this embodiment and embodiment 1 is that the end face of the sample injection needle 41 is conical with a diameter of 1.6 mm, a taper of 60 degrees, and a surface finish of Ra 9.8. In addition, in step a of the sample injection method, the movement speed of the sample injection needle 41 when leaving the dNTP reagent solution is 5 cm/s, and in step b, the movement speed of the sample injection needle 41 when leaving the sequencing reaction solution is 4.5 cm/s. The sample injection needle 41 moves up and down in the sequencing reaction solution 12 times before leaving the sequencing reaction solution.

The sequencing spectra and other experimental results (sequencing time, repeatability, and uniformity) obtained in this example were consistent with the data obtained in Example 1 within the theoretical error range, and the repeatability was 95%.

Finally, it is necessary to point out that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and are not to be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above contents of the present invention fall within the scope of protection of the present invention. Finally, it is necessary to point out that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and are not to be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above contents of the present invention fall within the scope of protection of the present invention.

Claims (10)

1. A DNA sequencing device based on pyrosequencing, characterized in that it comprises a sample area, a reaction area, and a detection area; the sample area includes a separation disc (2) arranged in a centrifuge, the separation disc (2) including at least one DNA separation area, the separation area including a hollow filter column (21) with a filter membrane (22) inside, the filter membrane (22) being made of polyethylene microspheres with a pore size of 10 μm, containing DNA single strands with affinity linkers and DNA single strands without affinity linkers. A single chain is separated after filtration through the filter membrane (22); the reaction zone includes a sample rack (4), a dNTP reagent tank (7), and a reaction tank (3). Multiple sample needles (41) are fixedly mounted on the sample rack (4). The sample rack (4) reciprocates between the dNTP reagent tank (7) and the reaction tank (3) via a linear motion device. A rotating shaft (42) is located at the center of the sample rack (4). The rotating shaft (42) drives the sample rack (4) to rotate via a motor. 4) A lifting device is provided between the sample rack (4) and the rotating shaft (42). The sample rack (4) moves up and down along the rotating shaft (42) through the lifting device. The reagent tank (7) is provided with multiple slots (71). The reagent tank (7) is provided with a substrate supply unit. The substrate supply unit delivers four nucleic acid substrates of dNTPs, namely dATP, dCTP, dGTP, and dTTP, to the slots (71) through the delivery pipe (1). The reaction tank (3) is provided with multiple reaction positions (31). The reaction positions (31) extend to the detection area. The detection area includes a bioluminescence detector (5) and a rotating stage (51). The bioluminescence detector (5) is detachably installed on the rotating stage (51). The rotating stage (51) drives the bioluminescence detector (5) to rotate through a motor. The sample area, reaction area, and detection area are installed on the analysis table (1). The side of the analysis table (1) is provided with a robotic arm for operation. The linear motion device is fixed on the analysis table (1) through a mounting bracket (11). 2. The DNA sequencing device based on pyrosequencing according to claim 1, characterized in that a fixing seat (44) is provided at the upper end of the rotating shaft (42), and a bearing is provided between the fixing seat (44) and the rotating shaft (42). 3. The DNA sequencing device based on pyrosequencing according to claim 2, characterized in that the linear motion device includes a movable slide rail (45) fixed on the mounting frame (11) and a movable slider (43) fixed on the fixing seat (44). 4. The DNA sequencing device based on pyrosequencing according to claim 2, wherein the lifting device comprises a lifting slide rail (46) fixed to the rotating shaft (42) and a lifting slider (47) fixed to the sample rack (4). 5. The DNA sequencing device based on pyrosequencing according to claim 1, characterized in that the separation disk (2) is provided with a plurality of collection tubes (23), the filter column (21) is installed in the collection tubes (23), and the filter membrane (22) is located at one end of the filter column (21) or at a non-end of the filter column (21). 6. The DNA sequencing device based on pyrosequencing according to claim 5, characterized in that the collection tube (23) is provided with a detachable top cover (231), and the top cover (231) is connected to the collection tube (23) via a connecting strap (232). 7. The DNA sequencing device based on pyrosequencing according to claim 1, characterized in that the sample rack (4) is circular, the sample needles (41) are evenly distributed along the circumference, and the reaction sites (31) are evenly distributed on the reaction groove (3) along the circumference with the same diameter as the sample needles (41). 8. The DNA sequencing device based on pyrosequencing according to claim 7, wherein the reaction sites (31) are made of transparent material and the bioluminescent detector (5) is located within the circle enclosed by all the reaction sites (31). 9. The DNA sequencing device based on pyrosequencing according to claim 8, wherein the bioluminescent detector (5) is a CCD camera, and the rotating stage (51) is provided with a slot (511) for placing the CCD camera. 10. The DNA sequencing device based on pyrosequencing according to claim 1, wherein the reaction zone further includes a cleaning tank (8) and a drying tank (6) installed on the analysis station (1), wherein the cleaning tank (8) and the drying tank (6) are located between the dNTP reagent tank (7) and the reaction tank (3).