CN113308524A - Nucleic acid high-throughput sequencing method based on fluorescence resonance energy transfer - Google Patents
Nucleic acid high-throughput sequencing method based on fluorescence resonance energy transfer Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a nucleic acid high-throughput sequencing method based on fluorescence resonance energy transfer, which respectively modifies donor and acceptor fluorescent groups of the fluorescence resonance energy transfer on DNA polymerase and dNTP, adds another type of nucleic acid molecule to be detected or modified DNA polymerase after fixing a position, and then puts the modified dNTP into circulation in turn, changes the space distance between donor and acceptor by the movement of the DNA polymerase on a DNA chain when the dNTP participates in base synthesis, generates a continuously changed FRET signal, combines the input dNTP type to obtain the corresponding base type of the reaction, and determines the nucleic acid sequence by continuous multiple reactions. The invention can measure FRET signals of single molecule level, single clone molecular cluster and single molecule multicopy, can realize rapid detection and analysis of nucleic acid, has low requirements on nucleic acid concentration and total amount, and avoids amplification errors caused by high-cycle PCR.
Description
Technical Field
The invention relates to a high-throughput sequencing method, in particular to a nucleic acid high-throughput sequencing method based on fluorescence resonance energy transfer.
Background
FRET (fluorescence resonance energy transfer) refers to an energy transfer phenomenon that occurs between two fluorescent molecules that are close (1-10nm) apart. The emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor fluorophore to a certain extent, resulting in the fact that the excitation energy of the donor induces the acceptor to fluoresce, and the FRET efficiency is inversely proportional to the 6 th power of the donor-acceptor spatial distance, and is thus extremely sensitive. However, much research has focused on the structure and interaction of proteins and nucleic acid molecules, ignoring the potential for sequencing unknown nucleic acid strands for FRET-highly sensitive features.
The current high-throughput sequencing technology is represented by secondary sequencing of Illumina, four-color fluorescence labeled dNTP is generally adopted as a synthesis raw material, 3' -OH modifies a chemical protecting group, so that only one base can be extended in each round of synthesis reaction, and then a chemical reagent is required to be added to remove the protecting group of the dNTP. SMRT (single-molecule real-time sequencing technology) introduced by Pacbio adopts another dNTP fluorescence labeling mode, namely, a fluorescence molecule is labeled on a phosphate group, so that the fluorescence group on the former dNTP is cut off in the synthesis of a lower base. However, the fluorescence signal detected by a single base only lasts for a short period of time, so the SMRT technique must rely on a high sequencing depth to reduce the error rate of the raw data, which is costly. Meanwhile, the existing sequencing platform instrument is expensive, the multicolor fluorescence system is complex to operate, the sequencing data return time is long, and the instrument is not friendly to samples and experiments needing rapid detection. In addition, high nucleic acid concentration and total amount requirements limit some rare samples, and amplification errors can also be introduced if PCR is performed after pooling for nucleic acid concentrations and total amounts that meet sequencing requirements.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a nucleic acid high-throughput sequencing method based on fluorescence resonance energy transfer, which combines high-throughput sequencing with FRET to provide a novel sequencing method, depends on that FRET efficiency and 6 power of a donor-acceptor space distance are in inverse proportion, is more accurate in low-depth data expression compared with SMRT, and is easy to build in various laboratories.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a fluorescence resonance energy transfer-based nucleic acid high-throughput sequencing method, wherein donor and acceptor fluorophores of fluorescence resonance energy transfer are respectively modified on DNA polymerase and dNTP, the dNTPs comprise dATP, dTTP, dCTP and dGTP, after one of the nucleic acid molecule to be detected or the modified DNA polymerase is fixed in position, another one is added for incubation together to combine the nucleic acid to be detected and the DNA polymerase, then the modified dATP, dTTP, dCTP and dGTP are sequentially and circularly put into a single reaction system of dNTP in sequence in the incubated system, the change in the spatial distance between donor and acceptor by the movement of DNA polymerase on the DNA strand when dNTPs participate in base synthesis generates a continuously changing FRET signal, and combining the input dNTP types to obtain the corresponding base types of the reaction, and determining the nucleic acid sequence through continuous multiple reactions.
Preferably, the donor and acceptor fluorophores of FRET are modified on DNA polymerase and dNTP, respectively, the donor fluorophore may be modified on DNA polymerase, and the acceptor fluorophore may be modified on phosphate group of dNTP; alternatively, an acceptor fluorescent group may be modified in the DNA polymerase, and a donor fluorescent group may be modified in the phosphate group of the dNTP.
Wherein, the fixing position of the nucleic acid molecule to be detected can be realized by fixing DNA polymerase or fixing the nucleic acid molecule to be detected.
Further, the test nucleic acid or modified DNA polymerase may be immobilized by covalent or non-covalent interactions, including base-complementary pairing, protein-nucleic acid covalent modification, and biotin-streptavidin non-covalent binding.
In the invention, because the fluorescence of the donor is measured by an optical method, the donor needs to ensure that the amplitude of the fluorescence cannot be seen by naked eyes, and simultaneously, a reaction reagent or Wash Buffer needs to be pumped in by a syringe, the position of the nucleic acid molecule to be measured needs to be fixed in order to avoid the change of the fluorescence position caused by the fluid flow.
Further, the modified dNTPs according to dATP, dTTP, dCTP and dGTP are sequentially and cyclically added into the system in the above order, and the modified dNTPs are sequentially and cyclically added into the system after the combination of the nucleic acid to be detected and the DNA polymerase, and the unreacted dNTPs which are further added must be washed away with a buffer solution or chemically degraded before adding new dNTPs each time.
Wherein one of dATP, dTTP, dCTP and dGTP can be respectively modified by four acceptors with different emission spectra, only one donor is modified on polymerase, and the four acceptors have different spectral overlap to achieve continuous synthesis and continuous sequencing.
Preferably, the nucleic acid to be detected or the modified DNA polymerase is immobilized on a chip made of a rigid material or a material dispersed in a solution, the rigid material includes glass, polymethyl methacrylate, and polyamide, and the material dispersed in the solution includes magnetic beads and microspheres. For example, MagVigen magnetic beads use the non-covalent interaction of biotin-streptavidin, and Dynabeads magnetic beads use magnetic methods to separate capture.
Preferably, the chip comprises a micropore array and a nanopore array, and the median distance between the nucleic acid molecules to be detected or the DNA polymerase immobilized in the array is in the range of 0.1 μm-1 mm. In the prepared micropore and nanopore chip, the distance between nucleic acid molecules to be detected is determined by the size and the arrangement of pores, and the distance range is mainly limited by the requirements of half-wavelength limitation and high flux of visible light, namely the distance range cannot be too low or too high, and the distance range has high flux on the premise of accurate sequencing.
Wherein, the sequencing method can measure FRET signals between donor-acceptor at single molecule level, and can also measure FRET signals of single clone molecule cluster and single molecule multicopy.
The method combines FRET and high-throughput sequencing technology, improves the sequencing accuracy by the dNTP labeling mode in SMRT and the high sensitivity characteristic of FRET, relaxes the requirements on the concentration and the total amount of a sample to be loaded, and realizes the rapid sequencing analysis of the sample. This method is characterized in that donor and acceptor fluorophores for FRET are modified in DNA polymerase and dNTP, respectively, the spatial positions of DNA synthesis reaction are fixed, a reaction system containing one of dATP, dTTP, dCTP and dGTP is periodically introduced in a predetermined order, the change of FRET signal is brought by the change of the spatial distance between donor and acceptor when dNTP participates in base synthesis, the base type corresponding to the reaction is estimated by combining the kind of the reactant introduced at that time, and the composition of the nucleic acid sequence is determined by the arrangement of the base types obtained by multiple reactions.
Before applying this sequencing method, the donor-acceptor selection should be of sufficient duration, most commonly used today as anthocyanin, Atto and Alexa series, in addition to spectral overlap. Next, a donor (or acceptor) fluorescent molecule is modified on the DNA polymerase, and an acceptor (or donor) fluorescent molecule is modified on the phosphate group of the dNTP. This modification mode has the advantage that the fluorophore on the previous dNTP is detached from the complex before the next dNTP is added, thus avoiding the interference of the fluorescence signal, otherwise the fluorophore on the dNTP needs to be chemically cleaved off. In addition, with the development of fluorescent molecule pairs, the dNTPs of the method can be further modified by four acceptors with different emission spectra respectively, and only one donor with different degrees of spectral overlap with the four acceptors is modified on polymerase. Directly put into all reaction systems, because different dNTPs participate in synthesis to obtain different FRET signals, namely corresponding to four different bases, the sequencing speed is greatly improved.
The spatial position of the DNA synthesis reaction needs to be fixed during sequencing, and the sequencing can be realized by fixing the nucleic acid molecules to be detected or fixing DNA polymerase. For the immobilized nucleic acid molecule, a known sequence is modified on the surface of a chip or magnetic beads and the like to immobilize the nucleic acid molecule to be detected through base complementary pairing, and the immobilized nucleic acid molecule can be used as a primer. In addition, the molecules to be detected can be captured by utilizing the non-covalent binding action of biotin-streptavidin and the like, so that the same or even stronger immobilization action of covalent binding is achieved, and the template can not be washed away in the fluid injection process. For the immobilized DNA polymerase, specific binding of biotin-streptavidin may be chosen, but not limited to. The environment for sequencing can be selected from a chip made of a rigid material, such as glass, polymethyl methacrylate, polyamide, etc., or a material dispersed in a solution, such as magnetic beads, microspheres, etc. Not only ensures certain rigidity, but also adapts to optical instruments.
When base synthesis is carried out, dNTP approaches to DNA polymerase, the space distance between donor and acceptor becomes smaller, and FRET signal is strong; when the next dNTP to be introduced is also involved in base synthesis, the fluorophore on the previous base is detached, and the FRET value rapidly decreases until the next base synthesis. Therefore, no matter the choice of immobilized enzyme or immobilized sequence, it will affect the length of the reads obtained, and their main limitations are the hardware response and the fluorescence duration of the donor-acceptor.
The sequencing method can measure FRET signals between donor and acceptor at a single molecule level, and can also obtain a single clone molecular cluster and a single molecule multi-copy through an experimental method to amplify the FRET signals between the donor and the acceptor, which is favorable for sequencing accuracy and longer reading length acquisition. Before sequencing reaction, reaction reagents containing single dNTP, including dATP, dTTP, dCTP and dGTP, are prepared, injected in sequence and periodically, and before input, the input dNTP which is input in the previous step and is not reacted must be washed away by using a buffer solution or chemically degraded. Whichever dNTP participates in base synthesis, the trend and magnitude of the change in the space distance between donor and acceptor are similar, so that the change in FRET signal is approximate and shows regular change.
Even when the sequence to be tested contains a repetitive sequence, the sequencing method is still applicable. When a certain dNTP is continuously involved in the synthesis of several identical bases, the spatial distance between donor and acceptor changes continuously, the FRET value also changes regularly, and the number of times of signal change can be used to infer that several bases are synthesized. Sequencing is a high throughput format including microwell arrays, nanopore arrays, etc., with the median distance between immobilized molecules in the range of 0.1 μm to 1 mm. The existing mature chip is designed and manufactured with millions of pores, and the combination of the existing mature chip and the method can greatly improve the sequencing flux.
The invention provides a new sequencing principle by applying FRET to nucleic acid sequencing, the effect is very obvious, the high sensitivity of FRET ensures that higher accuracy can be obtained during sequencing, the method has different sequencing principles compared with the existing mature high-flux especially second-generation sequencing, and a dNTP marking strategy of free acceptor excision is applied, the method has excellent performance in the aspects of accuracy and flux, and the FRET-based sequencing platform saves the cost compared with the commercial second-generation platform, can be realized in a common laboratory, and is more widely applied.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention provides a high-throughput sequencing method with high accuracy by combining the FRET principle. Compared with the existing method, the method has the greatest characteristic that the sequencing accuracy is high by utilizing a high-sensitivity FRET technology and an acceptor excision-free dNTP labeling method. Meanwhile, the method has low requirements on the concentration and the total amount of nucleic acid, is more friendly to some rare samples, and avoids high-cycle PCR (polymerase chain reaction) for meeting the commercial sequencing requirement, thereby bringing amplification errors. The method can be configured under a mature FRET platform, so that the rapid detection and analysis of the sample can be realized in a laboratory. And is suitable for other reaction methods related to base extension and connection, and has good flexibility and strong universality. The method of the invention can measure FRET signals at single molecule level, and can also measure FRET signals of single clone molecular clusters and single molecule multiple copies, thereby realizing high-flux rapid and accurate sequencing of nucleic acid.
Drawings
FIG. 1 is the set-up of the sequencing platform in example 1;
FIG. 2 is a schematic diagram of sequencing after immobilization of a nucleic acid molecule to be tested and a partial characteristic signal of FRET extracted by a hidden Markov model in example 2;
FIG. 3 is a diagram showing a sequencing scheme and a hidden Markov model extraction of a part of a characteristic signal of FRET after DNA polymerase is immobilized in example 3.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1
Preparation and application of microwell arrays: a PDMS (polydimethylsiloxane) chip containing numerous microwells (2 μm × 2 μm × 2 μm) was prepared by soft lithography, and more than 50 ten thousand microwells were used per chip, and a high-throughput microwell array or nanopore array was prepared in a laboratory or custom-made by a company. The form of fixing the space position of the sequencing reaction can capture the nucleic acid molecule to be detected by chemically modifying the surface of the chip with a known sequence (complementary with a joint for building a library), and simultaneously can be used as a primer during synthesis. The method of biotin-streptavidin may also be used to immobilize DNA polymerase, as shown in FIG. 1. A nucleic acid sequence is designed according to a known sequence on the surface of a chip and an A tail added in PCR operation, DNA ligase is used for completing connection on unknown nucleic acid molecules to be detected, so that nucleic acid fragments after library construction can be complementary with a fixed sequence on the surface of the chip, then the nucleic acid fragments to be detected with a target size are screened by magnetic beads, the length of the fragments to be detected is controlled below 500bp in principle so as to prevent the fragments from falling off the surface, and the requirement can be met by an ultrasonic and enzymological method. The concentration of the nucleic acid to be detected after the library building is quantified by using the Qubit, and the concentration of the nucleic acid containing at most a single molecule in each well is calculated by using a Poisson distribution formula (as follows).
In this example, λ is the average number of nucleic acid molecules in a well, k is 0, 1, 2 …, and P (X ═ k) represents the probability that k nucleic acid molecules are contained in a well. When lambda is 0.1, about 90% of the pores are empty, and 9% of the single-molecule capture rate is adopted, so that the pores contain at most one molecule. The diluted nucleic acid to be detected is loaded into an injector connected with a polytetrafluoroethylene tube and is slowly injected into the chip by an injection pump, so that the nucleic acid molecules are gradually fixed in the holes. The unimolecule fixed in the hole is amplified into a monoclonal molecular cluster through a PCR process, and a FRET signal between donor and acceptor can be obviously enhanced. As shown in FIG. 1, the prepared sequencing chip is placed on the stage of a total internal reflection microscope, the reaction system injected into the sequencing chip is loaded by an injector connected with a polytetrafluoroethylene tube, the sample is injected from the inlet of the sequencing chip in FIG. 1, and the waste liquid is collected at the outlet. Alexa Fluor 555 acceptor fluorescent molecules are modified on phosphate groups of dNTPs (four kinds of dATP, dTTP, dCTP and dGTP), Alexa Fluor 647 donor fluorescent molecules are modified on DNA polymerase, and the reaction system formula and the Wash Buffer formula for cleaning are as follows. Excitation light for the donor fluorophore is generated by a laser emitter. DNA polymerase needs to be injected into the chip in advance and incubated for 2min at 60 ℃ to be combined with nucleic acid molecules to be detected, and then modified dNTP reaction systems are added.
Four reaction systems containing a single dNTP were prepared, including dATP, dTTP, dCTP and dGTP, loaded separately into 4-pronged 1mL syringes, and fluid flow was precisely controlled using syringe pumps. After the sequencing platform is built, the sequencing operation can be started, the reaction systems are sequentially injected into the chip in a circulating mode according to the sequence of dATP, dTTP, dCTP and dGTP, the volume of each injected reaction system is preferably more than 2 times of the total capacity of the chip, the reaction system is extended for 3s at 72 ℃ after micropores are completely infiltrated, and the dNTP which is injected before but does not react is thoroughly washed away by Wash Buffer which is at least 5 times of the total capacity of the chip before each reaction reagent is injected. When a certain dNTP participates in base synthesis, FRET signal undergoes an increase-decrease process. The subsequent experiment data utilizes a hidden Markov model to extract a FRET characteristic signal to obtain sequencing reads, and the sequence information of the original nucleic acid chain can be deduced.
In this example, the construction of a universal sequencing platform using FRET, the preparation of reaction reagents and Wash Buffer are described. And then pumping the reaction reagent and the Wash Buffer by using a precise injection pump so as to better control the flow rate of the fluid, prevent the chip from cracking due to the overhigh flow rate of manual pushing, and accurately control the total volume of the pumped fluid.
Example 2
PDMS (polydimethylsiloxane) chips containing numerous microwells (2 μm. times.2 μm) were prepared by soft lithography on the basis of the sequencing platform of example 1, with more than 50 ten thousand microwells per chip. The format for fixing the spatial position of DNA synthesis is selected to chemically modify the known sequence (TCGCCTAT) on the surface of the chip, each microwell modifying an infinite number of this known sequence. BCAT2 Gene fragment (284bp, NCBI Homo sapiens Gene ID:587) was amplified using genomic DNA of GM12878, 50 μ L system: premix Ex Taq HS 25. mu.L, DNA 50ng, forward and reverse primers 0.4. mu.M each (forward primer: 5'-GGAATCAGAGCCCACGAGT-3', reverse primer: 5'-TATCCTTGACCGCACGAC-3'), less than 50. mu.L were supplemented with enzyme-free water. The amplification procedure was: 5min at 95 ℃; (95 ℃ for 30s, 60 ℃ for 20s, 72 ℃ for 30s)30 cycles; 5min at 72 ℃; storing at 4 ℃. The PCR product is designed with a joint sequence (5 '-TCGCCTAT-3') according to the modified known sequence on the chip and the A tail added by Ex Taq DNA polymerase, joint connection is realized through DNA ligase, so that the nucleic acid segment to be detected can be complementary with the fixed sequence on the surface of the chip, and then the nucleic acid segment to be detected with the target size is screened through magnetic beads. Then, small fragments (such as primers, etc.) and genomic DNA are screened out by magnetic beads, and the library building process in this example is completed. In the case of lambda of 0.1, the diluted nucleic acid molecules to be tested immobilized single molecules in 9% of the microwells, and since countless known sequences were modified in the microwells, the immobilized single molecules could be amplified into monoclonal molecule clusters by PCR. Subsequently, the Alexa Fluor 647 donor fluorescent molecule is modified on Ex Taq DNA polymerase and pre-injected into the chip to be incubated at 60 ℃ for 2min, and combined with the nucleic acid molecule to be detected.
Alexa Fluor 555 acceptor fluorescent molecules are modified on phosphate groups of dNTPs, and a reaction reagent and a Wash Buffer are prepared according to the formula in example 1. Then using a syringe pump to pump the single dNTP reaction reagent according to the sequence of dATP, dTTP, dCTP and dGTP, the pumping volume can be controlled by the syringe pump, and with more than 2 times of the total capacity of the chip, completely infiltrating the micropore and then extending for 3s at 72 ℃, and circulating the pumping until the sequencing is completed. It should be noted that dNTPs previously dosed but not reacted must be thoroughly washed away with a Wash Buffer of at least 5 times the total chip capacity before each dosing of the reagents. The laser generator in the platform of the total internal reflection microscope is used for generating exciting light, the intensity of the emitted light is recorded by software, at the moment, the intensity of the emitted light of the donor and the acceptor is required to be recorded simultaneously, so that the FRET efficiency can be calculated, and the total internal reflection microscope is superior to a confocal microscope in this respect. FIG. 2 shows a sequencing diagram of a nucleic acid molecule to be detected immobilized on the surface of a chip, and the FRET efficiency is calculated from the obtained donor-acceptor emission light intensity by the following formula.
In the formula, E represents FRET efficiency, FacceptorAnd FdonorIndicating the recorded intensity of the emitted light from the acceptor and donor. After normalization, a FRET value change curve with time can be drawn as shown in the lower part of fig. 2, and a FRET characteristic signal is extracted by using a hidden markov model. From the FRET curve of FIG. 2, it can be deduced that the sequence of the nucleic acid molecule to be tested corresponding to the data in the graph is 5 '-GACAT-3', when the type of dNTP to be used is ATGTC when the change in FRET value is combined. Therefore, the complete BCAT2 gene fragment can be repeatedly measured, and the accuracy of the sequencing result reaches over 99.9 percent according to the comparison of the existing gene sequences.
In this example, on the basis of example 1, the micro-well array chip and the monoclonal molecular cluster are used to improve the sequencing throughput and accuracy. The nucleic acid molecule to be detected is fixed on the surface of the chip, and the DNA polymerase modified with the donor molecule is combined with the template and the primer. Using the PCR product of the housekeeping gene as the nucleic acid molecule to be detected of the examples, the composition of the sequence can be accurately determined in the above sequencing step.
Example 3
Based on the sequencing platform of example 1, a nanopore array chip with millions of diameters of tens of nanometers, a thickness of 100nm, similar to a zero mode waveguide aperture in SMRT, is purchased or prepared from a chip company by spin-coating an electron beam photoresist on the surface of a clean substrate, forming a glue column corresponding to a desired aperture on the substrate after electron beam lithography, depositing gold metal, and then stripping, forming a nanopore at the position of the glue column. Modifying Alexa Fluor 647 donor fluorescent molecules and streptavidin on Ex Taq DNA polymerase, functionalizing the nanopore array chip biotin, and anchoring the DNA polymerase at the bottom of a nanopore through biotin-streptavidin non-covalent binding. Amplification of the BCAT2 gene fragment (284bp) using genomic DNA of GM12878, 50 μ L system: premix Ex Taq HS 25. mu.L, DNA 50ng, forward and reverse primers 0.4. mu.M each, and less than 50. mu.L were supplemented with enzyme-free water. The amplification procedure was: 5min at 95 ℃; (95 ℃ for 30s, 60 ℃ for 20s, 72 ℃ for 30s)30 cycles; 5min at 72 ℃; storing at 4 ℃. After the PCR product was purified, the diluted nucleic acid molecule to be detected and primers (forward primer: 5'-GGAATCAGAGCCCACGAGT-3', reverse primer: 5'-TATCCTTGACCGCACGAC-3') were put into the chip at a concentration of 0.1. lambda. and incubated at 60 ℃ for 2min to allow the single polymerase to bind to the DNA template and the sequencing primer, thus in this example, FRET measurement at the single molecule level was performed.
Due to the limitation of the nanopore, a long single DNA strand is clamped in the nanopore, and detection laser can be limited in the nanopore and cannot enter a solution area above the small hole, so that interference of FRET measurement at a single molecule level is avoided. Alexa Fluor 555 acceptor fluorescent molecules are modified on phosphate groups of dNTPs, and a reaction reagent and a Wash Buffer are prepared according to the formula in example 1. Then using a syringe pump to pump the single dNTP reaction reagent according to the sequence of dATP, dTTP, dCTP and dGTP, the pumping volume can be controlled by the syringe pump, and with more than 2 times of the total capacity of the chip, after the complete infiltration 72 ℃ extension 3s, pump in until the sequencing is completed. It should be noted that dNTPs previously dosed but not reacted must be thoroughly washed away with a Wash Buffer of at least 5 times the total chip capacity before each dosing of the reagents. The laser generator in the platform of the total internal reflection microscope is used for generating exciting light, the intensity of the emitted light is recorded by software, at the moment, the intensity of the emitted light of the donor and the acceptor is required to be recorded simultaneously, so that the FRET efficiency can be calculated, and the total internal reflection microscope is superior to a confocal microscope in this respect. When base synthesis occurs, the space distance between donor and acceptor is reduced, the FRET value is obviously increased, and then when next dNTP participates in synthesis, the acceptor group on the previous base is cut off, so that the subsequent sequencing process is not influenced. FIG. 3 shows a schematic diagram of sequencing after anchoring DNA polymerase at the bottom of the nanopore, and the obtained donor-acceptor emission light intensity is used to calculate FRET efficiency by the following formula.
In the formula, E represents FRET efficiency, FacceptorAnd FdonorIndicating the recorded intensities of emitted light from the acceptor and donor. After normalization, a change curve of a FRET value with time can be drawn, as shown in the lower part of FIG. 3, relative FRET efficiency is calculated according to the intensity of emitted light, and a characteristic signal of FRET is extracted by using a hidden Markov model. From the FRET curve of FIG. 3, it can be deduced that the sequence of the nucleic acid molecule to be tested corresponding to the data in the graph is 5 '-TGACAT-3', when the type of dNTP to be used in the change of the FRET value is ATGTCA. Therefore, the complete BCAT2 gene fragment can be repeatedly measured, and the accuracy of the sequencing result reaches over 99.9 percent according to the comparison of the existing gene sequences.
In this example, a nanopore array chip similar to a zero mode waveguide pore is prepared by fixing DNA polymerase, and the sequencing process is completed based on FRET principle, so that the sequence composition can be accurately measured in the above sequencing steps, and the flexibility and the universality of the sequencing method can be seen. The read length at this point is limited by the hardware response and the duration of the molecule to fluorescence.
In the embodiment 2 of the invention, the micropore array is used, the DNA synthesis position is fixed by utilizing the base complementary pairing effect between nucleic acid and molecules to be detected, and the signal of a monoclonal molecule cluster is detected, and the multiple copies of a single molecule are similar to the monoclonal molecule cluster; in example 3, the nanopore array is used for immobilizing DNA polymerase by utilizing the non-covalent binding effect of biotin-streptavidin, and detecting a single-molecule signal; the method can ensure that the sequencing accuracy is well represented, all the embodiments are common operations in a laboratory, are equipped on the existing microscope platform in the laboratory, combine with mature FRET technology, have good applicability, provide an instant sequencer for the common laboratory, and save time cost and expense for sending to a company for sequencing. Meanwhile, in the embodiments 2 and 3, single molecules are distributed through Poisson distribution, so that the experimental requirement is low, the requirements on the concentration and the total amount of nucleic acid are low, and the amplification error caused by performing high-cycle PCR for meeting the commercial sequencing requirement is avoided.
Claims (9)
1. A nucleic acid high-throughput sequencing method based on fluorescence resonance energy transfer is characterized in that donor and acceptor fluorophores of fluorescence resonance energy transfer are respectively modified on DNA polymerase and dNTP, wherein the dNTP comprises dATP, dTTP, dCTP and dGTP; adding another one after the nucleic acid molecule to be detected or the modified DNA polymerase is fixed in position, incubating to combine the nucleic acid to be detected and the modified DNA polymerase, adding the modified dATP, dTTP, dCTP and dGTP circularly in sequence, changing the space distance between a donor and an acceptor by the movement of the DNA polymerase on a DNA chain when the dNTP participates in base synthesis, generating a continuously changed FRET signal, combining the added dNTP types to obtain the base type corresponding to the reaction, and determining the nucleic acid sequence by continuous multiple reactions.
2. The fluorescence resonance energy transfer-based nucleic acid high-throughput sequencing method according to claim 1, wherein the donor and acceptor fluorophores for fluorescence resonance energy transfer are modified on DNA polymerase and dNTP respectively, and the donor fluorophore is modified on DNA polymerase, and the acceptor fluorophore is modified on phosphate group of dNTP; alternatively, an acceptor fluorescent group may be modified in the DNA polymerase, and a donor fluorescent group may be modified in the phosphate group of the dNTP.
3. The fluorescence resonance energy transfer-based nucleic acid high-throughput sequencing method according to claim 1, wherein the fixing position of the nucleic acid molecule to be detected is realized by fixing DNA polymerase or fixing the nucleic acid molecule to be detected.
4. The method for high-throughput sequencing of nucleic acids based on fluorescence resonance energy transfer according to claim 1, wherein the immobilization mode of the nucleic acid to be tested or the modified DNA polymerase is covalent interaction and non-covalent interaction, and preferably comprises base complementary pairing, protein-nucleic acid covalent modification or biotin-streptavidin non-covalent binding.
5. The method for high-throughput sequencing of nucleic acids by fluorescence resonance energy transfer according to claim 1, wherein the modified dATP, dTTP, dCTP and dGTP are sequentially and cyclically added into the system in which the modified dNTPs are sequentially and cyclically added into the system after the nucleic acid to be tested is combined with the DNA polymerase, and the unreacted dNTPs which are added in the previous step must be washed away with a buffer or chemically degraded before each new dNTP is added.
6. The method for high-throughput sequencing of nucleic acids based on fluorescence resonance energy transfer according to claim 1, wherein dATP, dTTP, dCTP and dGTP are modified by four acceptors with different emission spectra respectively, only one donor is modified by polymerase, and the four acceptors are overlapped with different spectra to a certain extent, so as to realize continuous synthesis and continuous sequencing.
7. The fluorescence resonance energy transfer-based nucleic acid high-throughput sequencing method according to claim 1, wherein the target nucleic acid or the modified DNA polymerase is immobilized on a chip made of a rigid material or on a material dispersed in a solution, the rigid material comprises glass, polymethyl methacrylate and polyamide, and the material dispersed in the solution comprises magnetic beads and microspheres.
8. The fluorescence resonance energy transfer-based nucleic acid high-throughput sequencing method according to claim 7, wherein the chip comprises a micropore array or a nanopore array, and the median distance between the nucleic acid molecules to be detected or the DNA polymerase immobilized in the array is in the range of 0.1 μm to 1 mm.
9. The fluorescence resonance energy transfer-based nucleic acid high-throughput sequencing method according to claim 1, wherein the sequencing method can measure FRET signals between donor-acceptor at a single-molecule level, and can also measure FRET signals of single-clone molecular clusters and single-molecule multiple copies.
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| CN109562376A (en) * | 2016-04-04 | 2019-04-02 | 纽约哥伦比亚大学董事会 | A kind of unimolecule based on fluorescence energy transfer/cluster DNA synthesis order-checking |
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| CN109562376A (en) * | 2016-04-04 | 2019-04-02 | 纽约哥伦比亚大学董事会 | A kind of unimolecule based on fluorescence energy transfer/cluster DNA synthesis order-checking |
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