CN114196737A

CN114196737A - Sequencing method for constant-temperature amplification

Info

Publication number: CN114196737A
Application number: CN202010984817.2A
Authority: CN
Inventors: 康力; 张晓璐; 乔朔; 孙文婷; 陈子天; 段海峰
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2022-03-18

Abstract

The application discloses a sequencing method for isothermal amplification. Based on the constant temperature amplification reaction which is modified by two primer microspheres and can occur in a test tube. The invention aims to provide a constant-temperature amplification technology which is carried out in a test tube, is quick and efficient, does not need a special and complicated library building process, does not need a separate amplification instrument, is compatible with sequencing methods such as fluorescence generation and the like, amplifies the surface of microspheres through a built library, and immobilizes the microspheres into a gene sequencing chip for sequencing.

Description

Sequencing method for constant-temperature amplification

Technical Field

The invention relates to a sequencing method for isothermal amplification, belonging to the field of gene sequencing.

Background

The high-throughput sequencing technology is one of the common analysis technologies in the field of life science at present, and can sequence DNA sample fragments to obtain DNA sequences of corresponding samples and establish the connection between biological phenotypes and genome, transcriptome and other levels. Second generation sequencing technologies have now occupied a significant portion of the market in the sequencing field because of their large throughput and relatively inexpensive price. The second generation sequencing technology is characterized in that a single DNA is not sequenced, but a DNA is amplified by thousands of times and then sequenced and signal acquisition are carried out, and different DNA amplification technologies are used. At present, the mainstream sequencing technology in the world has a corresponding specific amplification technology. The Illumina reversible end-termination sequencing technology uses a bridge amplification method: two different primers are planted on the surface of the planar chip, and then bridge PCR is carried out to obtain a DNA cluster; the semiconductor sequencing method of ion torrent corresponds to the amplification method of emulsion PCR: carrying out solid phase PCR on the microspheres in the formed emulsion, and then carrying the microspheres on a chip; the huada sequencer is an amplification mode of the used DNA nanospheres: the DNA is immobilized on the surface of the chip and amplified into a cluster of DNA nanospheres. However, these methods are either directly incompatible with existing fluorogenic sequencing methods, require either special and complex library construction procedures, or require additional complex instrumentation for amplification alone.

The invention discloses a constant temperature amplification method in a test tube, which is compatible with fluorogenic sequencing and based on two immobilized primer microspheres. The invention aims to provide a constant-temperature amplification technology which is carried out in a test tube, is rapid and efficient, does not need a special and complex library building process, does not need a separate amplification instrument, and is compatible with fluorogenic sequencing.

Disclosure of Invention

The invention aims to provide a sequencing method for isothermal amplification, aiming at the defects in the prior art, the isothermal amplification is carried out in the environment such as a test tube, and then microspheres are immobilized on a chip for sequencing.

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

the invention discloses a sequencing method of constant temperature amplification, which is characterized by comprising the following steps,

the microsphere and the amplification template form a mixed solution, two constant temperature amplification primers, namely a first amplification primer and a second amplification primer, are arranged on the microsphere, and a sequence of at least one amplification primer contains a site which can be cut;

hybridizing an amplification template to the microspheres, wherein the two ends of the amplification template contain common adaptor sequences, namely an adaptor sequence 1 and an adaptor sequence 2, and the adaptor sequence 1 of the amplification template hybridizes with a first amplification primer on the microspheres;

adding a reaction solution containing DNA polymerase, and extending the amplification primer under the action of the polymerase to form a DNA chain which is complementarily paired with the amplification template;

unwinding, and cleaning the amplified template fragment under unwinding to obtain a DNA chain with complementary pairing with the amplified template on the surface of the microsphere;

adding a constant-temperature amplification reagent, and carrying out microsphere surface amplification in the presence of a recombinase;

adding a shearing reagent, and shearing the template amplified by the modified primer into two sections;

adding a blocking reagent, and blocking the 3' end of the DNA on the surface of the microsphere;

immobilizing the microspheres on a gene sequencing chip by utilizing immobilization groups on the microspheres;

hybridizing the sequencing primer;

sequencing;

wherein the reaction solution containing the DNA polymerase contains the DNA polymerase and dNTP;

the adaptor sequence 1 and the first amplification primer are complementary paired, at least in part.

According to a preferred embodiment, the microsphere is modified with two kinds of amplification primers and is connected with an immobilization group; the two ends of the amplification template contain a common joint sequence, and the joint sequence 1 and at least partial sequence of the first amplification primer are in complementary pairing; the adaptor sequence 2 and the second amplification primer are at least partially sequence identical.

the method comprises the following steps that (1) a mixed solution is formed by polymer microspheres and an amplification template, wherein the polymer microspheres are provided with two constant-temperature amplification primers, namely a first amplification primer and a second amplification primer;

hybridizing the sequencing primer;

sequencing;

According to a preferred embodiment, preferably, the amplification is isothermal amplification.

According to a preferred embodiment, the amplification is one of RPA, RAA, bridge amplification.

According to a preferred embodiment, the time for the amplification of the RPA is between 5 and 90 minutes, preferably between 6 and 40 minutes, more preferably between 8 and 30 minutes, more preferably between 10 and 25 minutes.

According to a preferred embodiment, the amplification primers are 20-45bp in length.

According to a preferred embodiment, the microspheres have biotin as an immobilization group, and the gene sequencing chip has modified streptavidin thereon; the microspheres are attached to a gene sequencing chip by a specific reaction of biotin and streptavidin.

According to a preferred embodiment, the microspheres have a diameter of 0.3 to 5 microns, preferably 0.5 to 4 microns, more preferably 1 to 2 microns.

The invention also discloses a gene sequencing method, which is characterized in that a DNA molecule to be detected is broken into 50-1000bp segments which are used as amplification templates, and sequencing is carried out according to any one of the methods.

Has the advantages that:

the invention adopts a constant temperature amplification method in a test tube based on microspheres immobilized with two primers. By controlling the ratio of microspheres to template, the DNA clusters on a plurality of microspheres can be amplified by the same template. The amplified microspheres are immobilized on a high-throughput sequencing chip, so that the effect of being compatible with high-throughput sequencing can be realized. Compared with the amplification methods of companies such as illumina, ion torrent and the like, the method is rapid and efficient, does not need a special and complex library building process, and does not have a complex temperature control process of PCR and a complex fluid device for emulsion amplification. The technology of the invention can also be applied to other sequencing technologies such as reversible terminal termination sequencing of illumina, semiconductor sequencing of ion torrent and the like. (1) The invention directly carries out constant temperature amplification on the microspheres immobilized with two primers, and the reaction can be directly carried out in containers such as a test tube, an EP tube and the like. (2) The invention can control the number of microspheres with amplification products by controlling the density of the microspheres and the concentration of the template in the reaction solution, and the flux is controllable. (3) The invention uses a constant temperature amplification technology, which is faster and more efficient, has lower requirements on the device, and does not need a complex temperature control flow of PCR and a complex fluid device of emulsion amplification. (4) The invention does not need special and complex library building process, only needs to connect the connectors at the two ends of the DNA sample, and has more convenient library building process and shorter operation time. (5) The amplified microspheres can be subsequently immobilized on the surface of the chip, so that the method is compatible with a fluorescence sequencing technology, and the distinguishing modification inside and outside a chip pit is not damaged, so that the liquid seal during subsequent sequencing is not influenced. (6) The invention is compatible with other high-throughput sequencing technologies and has stronger compatibility.

Drawings

FIG. 1 is a flow chart of amplification.

Detailed Description

The gene sequencing chip of the present invention refers to a general chip having a fluid inlet and outlet and a reaction chamber. Similar gene sequencing chips have been disclosed several times in previous patents of the applicant, such as patents CN2017105741742, CN201710630287X, the contents of which may be incorporated by reference into this patent, if necessary.

The chemical ligation of the microspheres to the gene sequencing chip can be performed in a variety of ways. In general, it is preferred to use two chemical groups which are reactive with each other for the attachment. In particular, the microspheres can be fixed in a physical clamping manner, but the actual operation is difficult, and the requirement on the mutual matching of the microspheres and the bottom plate is higher.

Chemical ligation is a common conceivable method, and typically, click chemistry is used for ligation, e.g., one with an alkynyl group on one surface and an azide group on the other surface. Generally speaking, chemical ligation methods are more common, and different ligation methods can be selected according to different application environments. And will not be discussed again here.

The RPA is a constant-temperature amplification reaction at 37-42 ℃, and the reaction mainly depends on key proteins such as DNA polymerase, recombinase, single-strand binding protein and the like. The primer will first bind to the recombinase to form a primer-recombinase fiber before binding to the complementary sequence. In the reaction, the primer opens the double-stranded DNA as a template by the action of the recombinase and its associated coenzyme, and performs strand exchange, binding to the opened single strand at the complementary pair. Subsequently, the single-chain binding protein will bind to the opened single chain and maintain the single-chain structure. Then, the primer unwinds the double strand edge under the action of the DNA polymerase having the 5 '-3' strand substitution activity to synthesize a new strand, and finally forms a new DNA double strand and a substituted DNA single strand. The primers at both ends are subjected to the above reaction under the action of enzyme, so that the template can be subjected to exponential amplification

And (3) in an RPA amplification cycle, during amplification, the primer is combined with recombinase and combined with the complementary pairing region under the action of the recombinase to form a D-type stem-loop structure. Then the single-strand binding protein is combined with the uncoiled single strand, and the combined primer is extended under the action of DNA polymerase to form a new double strand and uncoiled single strand, thus completing the amplification process. During the formation of the primer-recombinase polymer, when ATP is hydrolyzed, the primer-recombinase polymer is disintegrated and the recombinase uvsX is replaced by the single-stranded binding protein gp 32. In the presence of the auxiliary binding factor uvsY and the macromolecule Carbowax 20M, the equilibrium is shifted toward the direction of generating the primer-recombinase.

More critical in the RPA reaction is the formation of the primer-recombinase fiber by ATP and the recombinase uvsX. When ATP is hydrolyzed, the primer-recombinase polymer will break down and the recombinase uvsX will be replaced by the single-stranded binding protein gp 32. In the presence of the auxiliary binding factor uvsY and the macromolecule Carbowax 20M, the equilibrium is shifted toward the direction of generating the primer-recombinase.

The RPA technique was first reported in 2006 by pinenburg et al (pinenburg et al, 2006) and fluorescence quantification of DNA was accomplished using this technique. Subsequently, the twist dx company to Piepenburg introduced a twist amp amplification kit based on RPA amplification technology. In 2017, twist dx continues to provide a new liquid RPA kit. In 2018, the twist Dx company provided a new RPA detection means by utilizing formamidopyrimidine-DNA glycosylase, and the direct detection of RPA on a test strip is realized.

The RPA amplification technology is rapid in reaction, generally the whole reaction only needs 5-20 minutes, and finally the template can be amplified by 1011-1012 times. The amplification primer of the RPA is different from the PCR primer, the length of the general primer is about 30nt, the optimal length of the amplified template is 100-200 bp, the template of 80-400 bp can be amplified, the longest template of 1.5kb can be amplified, but the amplification effect is obviously influenced. In addition, RPA amplification has good sequence specificity, can amplify different target sequences, and amplification is not affected by a plurality of PCR inhibitors (Lillis et al, 2016; Lobato and O' Sullivan, 2018; Piepenburg et al, 2006). Due to its excellent amplification rate and amplification specificity, RPA technology is often used in the field of liquid biopsy and the like.

The isothermal amplification described in the present invention may be RPA or RAA. Two primers were involved in RPA recombinase polymerase amplification. Generally, three enzymes are involved, recombinase, single-stranded DNA binding protein, polymerase. More information about the RPA may be queried for the apendix to the twistamp interaction kit.

The isothermal amplification of the invention can be carried out in containers such as EP tubes, and after the amplification is finished, microspheres are fixed on a gene sequencing chip and then sequencing is carried out.

A sequencing method of isothermal amplification is characterized by comprising the following steps,

the method comprises the following steps that (1) a mixed solution is formed by polymer microspheres and an amplification template, wherein the polymer microspheres are provided with two amplification primers, namely a first amplification primer and a second amplification primer, and one or two primer sequences contain special sites capable of being cut;

hybridizing an amplification template to the microspheres, wherein the two ends of the amplification template contain a common linker sequence, the linker sequence is matched with two amplification primers on the surfaces of the microspheres, the amplification template is complementarily matched with the primers on the microspheres through the linker sequence, and the hybridization of the template is completed after the hybridization process of the template, (the efficiency of the hybridization step is related to the temperature process and the solution components during the hybridization of the template, and the solution components are generally high-concentration salt solutions, such as 5XSSC buffer solution and the like);

unwinding, and cleaning the unwinding amplified template fragment with the adaptor sequence to obtain a DNA chain with complementary pairing with the amplified template on the microsphere surface;

adding a constant-temperature amplification reagent for amplification, in the presence of recombinase, complementarily pairing the amplification primer with the other end of the DNA template extended from the solid phase and further extending a new template, and circulating the steps so as to amplify the template on the surface of the microsphere;

after the microspheres are centrifugally cleaned, adding a shearing reagent, shearing the amplified solid-phase double-stranded template into a single strand, and hybridizing a subsequent sequencing primer;

after the microspheres are centrifugally cleaned, adding a blocking reagent, and blocking the 3' ends of all solid-phase DNA;

hybridizing the sequencing primer;

sequencing;

wherein the reaction solution contains dNTP;

hybridizing the amplification template to the microsphere, namely hybridizing the amplification template and two amplification primers together in a base complementary pairing mode;

the other end of the DNA chain is connected to the microsphere, and the other end of the amplification template is hybridized with the other solid phase primer.

According to a preferred embodiment, two kinds of amplification primers are modified on the polymer microsphere, and an immobilization group is connected to the polymer microsphere; the amplification template contains a common linker sequence at both ends, which is complementary paired to the amplification primers.

According to a preferred embodiment, the microspheres are one of inorganic material microspheres or organic polymer microspheres.

According to a preferred embodiment, the microspheres are one of silica microspheres, PS microspheres, hydrogel microspheres.

According to a preferred embodiment, the microspheres are composite microspheres.

The invention provides a sequencing method of isothermal amplification, which is characterized by comprising the following steps,

mixing polymer microspheres containing immobilized groups with an amplification template; the polymer microsphere is modified with two kinds of amplification primers, namely a first amplification primer and a second amplification primer;

hybridizing the amplified template to the surface of the microsphere; both ends of the amplified template contain a common linker sequence, and the sequence is complementary and matched with the amplification primer of the microsphere; adding reaction liquid containing DNA polymerase to react to form a DNA chain which is complementarily matched with the amplification template;

washing, adding DNA helicase solution for reaction, and washing the helicase fragments;

adding an amplification reagent for amplification;

hybridizing the sequencing primer;

and (5) sequencing.

According to a preferred embodiment: preferably, the amplification is RPA, RAA, bridge amplification.

According to a preferred embodiment, the time for the RPA amplification is between 5 and 90 minutes.

The invention also provides a gene sequencing method, which is characterized in that the DNA molecule to be detected is broken into 50-1000bp segments which are used as amplification templates, and sequencing is carried out according to the method.

Primers are often used in amplification reactions. The present invention relates to at least two amplification primers. The two primers serve not only to pair with the template binding, but also to attach the other end of the DNA strand to the microsphere in the presence of a recombinase. Thus, during the amplification process, the amplification process on the surface of the microsphere is formed. There is no special requirement for the design and synthesis of primers. The two primer sequences involved in the invention are respectively:

primer 1(39 bp): 5' -dUGAAGGTGTGCCATCTCATCCCTGCGTTTCCCGAGTCAG;

primer 2(41 bp): 5' -CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT.

The primer 1 and the primer 2 are one example. The requirements for the number of primers, etc., for different amplification reactions vary. The isothermal amplification reaction involved in the present invention may use at least two types of primers. The specific design of the primers can vary.

The template in the invention refers to a DNA fragment to be detected, and can also be called an amplification template. The amplification template is the fragment to be amplified. The sequence of the template may be unknown. As can be seen from the above statement, the sequencing process can be performed after the long fragment of DNA is broken into small fragments and then the linker sequence is ligated. In the whole process, there is no limitation on the DNA fragment or nucleic acid sequence fragment to be detected, and any sequence can be used for the operation. This is also the concept of complete sequencing.

The gene sequencing chip provided by the invention refers to a high-flux gene sequencing chip, and is a common second-generation gene sequencing chip. The applicant previously filed a number of patents on gene sequencing chips. It should be noted that the gene sequencing chip of the present invention is not limited to any one chip. The gene sequencing chip of the invention needs to contain a micro-pit with a proper size. The appropriate size refers to a micro-pit matching the size of the microsphere. For example, 10% larger than the diameter of the microsphere, for example, 20% larger than the diameter of the microsphere, for example, 30% larger than the diameter of the microsphere, for example, 40% larger than the diameter of the microsphere, for example, 50% larger than the diameter of the microsphere, for example, 100% larger than the diameter of the microsphere; any range of intervals wherein any two ratios make up may be used.

The size of the micro-pits is 0.2-10 microns, preferably 0.25-5 microns, more preferably 0.3-3 microns, more preferably 0.5-2 microns. The micro-pits refer to regularly arranged micro-pits. Generally, on at least one inner surface of a gene sequencing chip, there are a large number of regularly arranged micro-pits for high-throughput sequencing. The intersection of the micro-pits and the inner surface of the chip is formed into a circular shape or an approximate circular shape. The micro-pits are not required to be in strict cylindrical shapes, and due to various micro-nano processing means, the processed micro-pits have various shapes, most commonly are approximate to elliptical micro-pits and can also be close to a cubic column, but no sharp micro-pits exist in the corner area. The shape of the micro-pits is more of an optical effect of actual sequencing, and the effect on the reaction is not very large.

When sequencing genes, a small amount of data does not have much significance. Specific next generation gene sequencing refers to high throughput sequencing. Sequencing of a few fragments is not of practical industrial value. Therefore, the size of the micro-pits is generally very small. And has the distinct characteristic that no special operation for a single micro-pit exists in the whole sequencing process. Typically experimental results or data interpretation of a large number of micro-pits.

The micro-pits in the invention refer to the microstructure of a common sequencing chip, generally, the structure has concave-convex performance, and the concave-convex structure is similar to a circle or an ellipse or a square. Each small micro-pit is a reaction chamber, and when fed back to the sequencing data, it corresponds to a data point. This is also a common fact in sequencing reactions.

Sequencing as referred to in the present invention refers to secondary sequencing.

The gene sequencing chip of the invention refers to a sealed gene sequencing chip. Comprises a fluid inlet and outlet, a reaction chamber and a micro-pit on the side wall of the reaction chamber. The microspheres are immobilized in the micro-pits by chemical or physical means. Each micro-pit is a reaction unit. When the gene is sequenced, the sequence of the gene is judged by judging the signal in each micro-pit.

A primer refers to a molecule having a specific nucleotide sequence, which stimulates the synthesis of a macromolecule having the specific nucleotide sequence at the start of nucleotide polymerization, and which is covalently linked to a reactant, and is referred to as a primer. The primers are typically two oligonucleotide sequences synthesized by man, one complementary to one of the DNA template strands at one end of the target region and the other complementary to the other DNA template strand at the other end of the target region, and function as the initiation point for nucleotide polymerization, from which 3 ends the nucleic acid polymerase can begin synthesizing new nucleic acid strands. Primers designed artificially in vitro are widely used for polymerase chain reaction, sequencing, probe synthesis, and the like. The invention relates to an amplification primer and a sequencing primer. The design of primers is a common technique. In the prior art, many researches are carried out, and the invention is not described in detail.

Gene sequencing is a process of base determination. In conventional gene sequencing, especially meaningful next-generation gene sequencing is generally a process of converting a chemical signal into an optical signal or other signal. Generally, the optical signal of a single molecule is difficult to detect, and the extremely sensitive CCD cannot distinguish, so that the fragment to be sequenced needs to be copied in many copies by an amplification method, and in combination with the microsphere amplification technology in the present application, there is generally only one fragment to be detected on the same microsphere, and many copies are copied by an amplification method, so that the reaction signal corresponding to the base can be detected by a device such as CCD during the subsequent chemical reaction. In this process, preliminary preparation is important. The results of different amplification methods are somewhat different, for example, the warfarin uses a bridge amplification method, which amplifies to obtain a globular-like structure. In the present application, the isothermal amplification of short fragments on microspheres is used to obtain non-spherical amplification results linked to microspheres.

Overall, the results of a strict comparative isothermal amplification are not of much interest. The applicant previously filed several patents on sequencing methods or techniques, such as CN201510155218.9, CN201510212788.7, CN201510212789.1, CN201510822361.9, cn201510815685.x, CN201510944878.5, cn201610899880.x, etc., which are multi-base sequencing methods belonging to fluorescence switching. The contents of the above patents may be incorporated by reference into this patent, where necessary.

According to a preferred embodiment, the sequencing is a 3-terminal unclosed sequencing.

the method comprises the following steps that (1) a mixed solution is formed by polymer microspheres and an amplification template, wherein the polymer microspheres are provided with two constant-temperature amplification primers, namely a first amplification primer and a second amplification primer, and at least one amplification primer sequence contains a site which can be cut;

adding a shearing reagent, and shearing the amplified double-chain template into a single chain;

hybridizing the sequencing primer;

sequencing;

wherein, the reaction solution containing the DNA polymerase contains the DNA polymerase, dNTP and corresponding reaction buffer solution;

According to a preferred embodiment, the amplification referred to in the present invention refers to amplification in the sense of the art. Amplification refers to gene amplification, which is the increase in the copy number of a gene by a certain technique.

According to a preferred embodiment, the adaptor sequence 2 and the second amplification primer are at least partially identical in sequence.

dNTP, deoxy-riboside triphosphate, is used for the reaction solution for amplification.

The blocking is a common step in solid-phase amplification, and the common blocking refers to a step of transferring a substrate modified at the 3 'end, such as ddNTP, to the 3' end of a solid-phase amplification product by using a tool enzyme, such as terminal transferase.

The shearing is a common step of molecular biology and surface amplification, and the common shearing refers to shearing bases and chemical sites with special modification by using tool enzymes such as endonuclease and the like, and finally dividing a certain solid phase primer extended template into two sections.

According to a preferred embodiment, the at least one amplification primer sequence comprises a cleavable site, meaning that one or both amplification primers comprise a cleavable site. When both amplification primers contain sites that can be cleaved, the cleavage sites of the two amplification primers should be different, and the conditions for cleavage should be different. Therefore, when the shearing is carried out, different shearing conditions are selected according to specific requirements, and the favorable shearing can be carried out in a controllable way.

Shearing and capping are common steps in the process of DNA editing. The shearing is a common step of molecular biology and surface amplification, and the common shearing refers to shearing bases and chemical sites with special modification by using tool enzymes such as endonuclease and the like, and finally dividing a certain solid phase primer extended template into two sections. The blocking is a common step in solid-phase amplification, and the common blocking refers to a step of transferring a substrate modified at the 3 'end, such as ddNTP, to the 3' end of a solid-phase amplification product by using a tool enzyme, such as terminal transferase.

The whole amplification process is shown in FIG. 1. Two solid phase primers are immobilized on the microspheres, one of which contains a cleavage site for the subsequent cleavage step (step 1). Before amplification begins, the template is first hybridized to the microspheres (step 2). Thereafter, initial extension (step 3), unwinding (step 4) and removal of the liquid phase template are performed. Then, amplification of the template was performed using the RPA kit (steps 5 and 6). After the RPA is completed, the cleavage reaction (step 7), the capping reaction (step 8) and the unwinding step (step 9) of the solid phase primer are performed in this order. Because the microspheres are also distributed in the solution, if the liquid-phase reaction solution and the template in the previous step are to be removed completely, the microspheres need to be centrifugally cleaned for several times. In addition, biotin carried on the microspheres can be bound to streptavidin on the surface of the chip, so that the microspheres are immobilized in the microwells of the chip. After amplification is finished, the template on the microsphere is single-stranded, and sequencing can be performed by hybridizing a sequencing primer to the microsphere after the microsphere is immobilized.

Herein, the two solid phase primers are also referred to as two solid phase amplification primers, or amplification primers, i.e. a first amplification primer and a second amplification primer.

The invention has two linker sequences, which can be connected to the two sides of the amplification template. Referred to as a first linker sequence and a second linker sequence.

In general, in sequencing, a DNA molecule of a fragment is in a double-stranded form, and before hybridization, the double-stranded molecule needs to be converted into a single-stranded molecule. The amplification template in the invention refers to a single-stranded DNA fragment at one end; and the linkers at both ends of the single-stranded fragment are referred to as linker sequence 1 and linker sequence 2.

In the present invention, the amplification template refers to an arbitrary DNA fragment sequence. In high throughput sequencing, many, e.g., more than 100M, DNA fragments at a time, any of which can be referred to as an amplification template.

According to the description of the present invention, the amplification templates contain common linker sequences at both ends, which means that the same linker sequences are connected to both ends of any amplification template. For example, two different linker sequences are ligated based on the difference between the 3-and 5-termini. Of course, in the case of complementary DNA strands, they may actually contain the complementary linker sequence of linker sequence 1 and the complementary linker sequence of linker sequence 2. This is within the routine knowledge in the art.

The adaptor sequence 1 and the first amplification primer are complementary paired, at least in part. Thus, when the amplification templates are hybridized, hybridization can be performed by means of complementary pairing.

The adaptor sequence 2 and at least part of the sequence of the second amplification primer are coincident. After the hybridization is completed, the reverse DNA strand of the amplified template, or the complementary paired strand, is obtained by unwinding, and in this case, the linker sequence is also reverse or complementary paired. Thus, the reverse adaptor sequence is required to bind to the second amplification primer in a subsequent isothermal amplification reaction. That is, the adaptor sequence 2 and at least a portion of the sequence of the second amplification primer are coincident. Of course, amplification primers generally need to meet other requirements for attachment to the microsphere, such as inclusion of groups attached to the microsphere, and the like. The requirement of partial matching is a common design approach.

For gene sequencing methods, it is conventional, for example, to measure one base per round for ILLUMINA. Most sequencing methods measure the signal of one base at a time. For the purposes of the present invention, it is not essential to react several bases at a time. The isothermal amplification method is more suitable for sequencing of 2+2 fluorescence switching type disclosed by the applicant before; but does not exclude conventional sequencing methods like the illumina type. In general, when applied to the 2+2 fluorescence switching (also known as fluorogenic sequencing) class of sequencing, the sequencing process matches due to the subsequent sequencing.

The biochemical compounds or modifications of the present invention are common in the art and commercially available.

Example 1

mal-PEG-biotin modification:

1) (a)0.45mM solution preparation: the powder was added to 1mg of mal-PEG-Biotin in 400uL 1XPBS to give 0.45 mM.

2) One chip was loaded with 200uL of solution. And (4) transferring 200uL of the solution by using a 200uL pipette, adding the solution into the washed gene sequencing chip, sealing the chip and reacting at normal temperature for 30 min.

2. Streptavidin (streptavidin) modification

1)1mg/mL streptavidin was diluted to 100ug/mL with 1 XPBS.

2) And (3) transferring 200uL of the pipette with 200uL of the pipette, adding the pipette into the chip from the sample adding port, sealing the chip, and reacting at normal temperature for 30 min.

3. Microspheres

The microspheres are selected in various ways, and the microspheres made of hydrogel, glass, PS and the like can be purchased according to specific requirements, and the surfaces of the microspheres can be modified by a common method.

The microspheres have a size range of 0.3 to 5 microns, preferably 0.5 to 4 microns, more preferably 1 to 2 microns in diameter, where 0.6,0.8,1.2,1.5 microns are optional ranges or extremes.

4. Immobilization of microsphere surface amplification primers

1) According to Table 1: the required amount of each material solution is added into the microsphere primer immobilization experiment material table, and the reaction is carried out for 4 hours at 65 ℃.

2) Cleaning microspheres: after the reaction is completed, the microspheres are centrifugally cleaned. The reacted 50mL centrifuge tube was removed, returned to room temperature, vortex for about 5s, added 20mL and 25mL solid carrier liquid (1 st time), placed in a centrifuge, and centrifuged at 16000g for 3 min.

3) And taking out a 50ML centrifuge tube, adding 20ML and 25ML of solid carrier liquid respectively, and centrifuging.

4) Storing in 4 degree refrigerator.

Wherein the alkynyl primer F has a sequence as follows: 5' -dUGAAGGTGTGCCATCTCATCCCTGCGTTTCCCGAGTCAG

Wherein the alkynyl primer R has the sequence: 5' -CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT

Alkynyl in both primers means that the 5' end of the primer is chemically modified to attach an alkynyl group.

Thus, when reacting with the nitrogen-nitrogen triple bond, they can be linked together by click chemistry.

TABLE 1 microsphere primer immobilization experiment material table

5. Template prehybridization

1)0.1M NaOH was used for unwinding. 1.5ml of DNA EP tubes are used for template dilution and unwinding; it can be diluted with ultrapure water.

Form panel	Final concentration of template	Template added and 0.1M NaOH volume
			1	0.2pM	8ul NaOH +8ul 10pM template
2	1pM	4ul NaOH +4ul 100pM template
			3	10pM	4ul NaOH +4ul 1nM template

TABLE 2

2) Adding 0.1M NaOH, shaking and mixing.

3) After the end of the unwinding, 5XSSC solution was added based on the volume of the unwinding liquid, resulting in a final volume of 400 ul.

4) 180ul of the template solution was taken out and placed in a PCR tube, and 20ul of 10M/ul of an aqueous microsphere solution was added thereto.

5) The annex program was selected on a PCR instrument: 96 ℃ for 30 s; -0.05 ℃/s; 10s at 40 ℃; 25 ℃ and forever.

6) After the reaction, the PCR tube was taken out and the microspheres were transferred into an EP tube.

7) The EP tube was placed in a centrifuge at 12000g for 3min, 195ul of the supernatant was removed leaving only about 5ul of the cleaned microsphere solution.

6. Initial extension reaction

1) 100ul of 2X high-fidelity DNA polymerase (Phusion) premix is placed on ice, then 100ul of ultrapure water is added, diluted by one time, shaken and mixed evenly, and placed on ice.

2) Adding the mixed solution into an EP tube, uniformly mixing the solution, transferring the mixed solution into a PCR tube, and heating the mixed solution on a PCR instrument at 72 ℃ for 2 min.

3) After the reaction, the PCR tube was taken out and the microspheres were transferred into an EP tube.

4) The EP tube was placed in a centrifuge at 12000g for 3min, 195ul of the supernatant was removed and about 5ul of the cleaned microsphere solution was retained.

7. Untwisting

1) 200ul of formamide were added to the EP tube and the reaction was carried out at room temperature (25 ℃ C.) for 10 min.

2) 195ul of the supernatant was removed and only about 5ul of the cleaned microsphere solution remained.

8. Recombinase polymerase reaction

1) The preparation of the RPA reaction solution was carried out according to the following Table 3.

Reagent	V/ul
		Hydration liquid	120
Magnesium acetate	10
		Ultrapure water	70
Total volume	200

TABLE 3

2) Adding the prepared and mixed amplification reaction solution into an EP tube, uniformly mixing the solution, transferring the solution into a PCR tube, placing the PCR tube on a PCR instrument, screwing down a cover, and selecting a heating program: 60min at 40 ℃; 4 ℃ and forever.

4) 195ul of the supernatant was removed and only about 5ul of the cleaned microsphere solution remained.

9. Shear fluid (USER enzyme mix) reaction

1) A1.5 ml DNA Lobind EP tube was used to prepare a U-cut reaction solution according to the following Table 4.

Reagent	V/ul
		USER enzyme	2
Cutsmart	20
		Ultrapure water	178
Total of	200

TABLE 4

2) After the preparation, the mixture is shaken and mixed evenly, quickly centrifuged for 2s, and placed on ice for standby.

3) Adding the prepared and mixed shearing reaction liquid into a chip, uniformly mixing the solution, transferring the mixed solution into a PCR tube, placing the PCR tube on a PCR instrument, and selecting a shearing liquid reaction program: 30min at 37 ℃; 4 ℃ and forever.

4) After the reaction, the PCR tube was taken out and the microspheres were transferred into an EP tube.

5) 195ul of the supernatant was removed and only about 5ul of the cleaned microsphere solution remained.

10. Reaction of end-capping solution (TdT enzyme mix)

1) A1.5 ml DNA Lobind EP tube was used to prepare a capping reaction solution according to the following Table 5.

Reagent	V/ul
		TdT enzyme	2
10 Xbuffer	20
		CoCl2	20
ddNTP(10mM，each)	4
		Ultrapure water	154
Total of	200

TABLE 5

3) Adding the prepared and mixed end-capping reaction solution into a chip, uniformly mixing the solution, transferring the solution into a PCR tube, placing the PCR tube on a PCR instrument, screwing down a cover, and selecting an end-capping solution reaction program: 30min at 37 ℃; 4 ℃ and forever.

11. Untwisting

12. Carrying microspheres:

1) 200ul of 1xPBS solution containing 0.01% Tween was added to the EP tube.

2) And (4) transferring the diluted microspheres by using a 200uL liquid transfer gun, aligning the microspheres to a sample injection port of the chip, and introducing the microspheres.

3) Centrifuging, wherein the bottom plate surface of the chip faces upwards. 1000rcf for 10 min.

13. Hybridization sequencing primer

1) The sequencing primer dissolved in 1 × TE at a concentration of 100uM was diluted to 2uM with hybridization solution in an EP tube.

2) After the preparation is finished, shaking and mixing uniformly.

3) Adding the prepared and mixed sequencing primer solution into a chip, placing the chip on a flat PCR instrument, screwing down a cover, and selecting a sequencing primer hybridization program: 7min at 60 ℃; at 40 ℃ for 3 min.

14. After the reaction was completed, 500uL of the solution was added and washed.

Example 1 discloses a specific example, detailing the entire procedure. The steps of which are fully supported for the claimed solution. Those skilled in the art will appreciate that certain numbers, such as amounts, heating times, specific models, etc., are within the routine skill of the art; are within the skill of those in the art to know the overall procedure. Likewise, the specific steps and materials can be selected by those skilled in the art based on the overall technology. It has to be mentioned, however, that the collocation of the overall steps, such as the solution described in claim 1, is of an overall design, which is of significant significance for the overall technical combination. While some knowledge is clearly within the routine skill of the art, a simple introduction and alignment is desired. The technical scheme of the invention belongs to the application of the field of isothermal amplification in gene sequencing. The field of gene sequencing is different from conventional detection, and has larger flux, requires more precision, and needs to consider more practical application values, rather than simply whether the method is available or not.

The above embodiments are preferred embodiments of the present invention, and those skilled in the art can make variations and modifications to the above embodiments, therefore, the present invention is not limited to the above embodiments, and any obvious improvements, substitutions or modifications made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. A sequencing method of isothermal amplification is characterized by comprising the following steps,

hybridizing the sequencing primer;

sequencing;

2. The method of claim 1, wherein: wherein two kinds of amplification primers are modified on the microsphere and are connected with an immobilization group; the two ends of the amplification template contain a common joint sequence, and the joint sequence 1 and at least partial sequence of the first amplification primer are in complementary pairing; the adaptor sequence 2 and the second amplification primer are at least partially sequence identical.

3. A sequencing method of isothermal amplification is characterized by comprising the following steps,

hybridizing the sequencing primer;

sequencing;

4. A method according to any one of claims 1-3, characterized in that: preferably, the amplification is isothermal amplification.

5. A method according to any one of claims 1-3, characterized in that: preferably, the amplification is one of RPA, RAA, bridge amplification.

6. The method of claim 5, wherein: the time for RPA amplification is 5-90 minutes.

7. The method of any one of claims 1 to 3, wherein the amplification primers are 20 to 45bp in length.

8. The method of any one of claims 1 to 3, wherein the microspheres have biotin as an immobilization group and the gene sequencing chip has modified streptavidin thereon; the microspheres are attached to a gene sequencing chip by a specific reaction of biotin and streptavidin.

9. The method according to any of claims 1 to 8, wherein the microspheres have a diameter of 0.3 to 5 microns, preferably 0.5 to 4 microns, more preferably 1 to 2 microns.

10. A method of gene sequencing, wherein a DNA molecule to be tested is fragmented into 50-1000bp fragments, which are used as amplification templates, and sequenced according to the method of any one of claims 1-9.