CN117701684A - Preparation method of gene chip - Google Patents
Preparation method of gene chip Download PDFInfo
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- CN117701684A CN117701684A CN202311745011.8A CN202311745011A CN117701684A CN 117701684 A CN117701684 A CN 117701684A CN 202311745011 A CN202311745011 A CN 202311745011A CN 117701684 A CN117701684 A CN 117701684A
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Abstract
The present application relates to a method for preparing a gene chip, comprising two stages: a first stage comprising providing a functional substrate formed with array spots of a probe precursor, a second stage for obtaining a first complete probe by introducing a first template molecule to extend on the probe precursor in the presence of a DNA polymerase and its reaction system to form a specific recognition component of the first target probe; and (3) carrying out inactivation and cleaning treatment to obtain the gene chip with the first complete probe single strand. The method can fully combine the advantages of industrial batch manufacturing of the gene chips with the diversified detection requirements of users, can accelerate the development progress of the gene chips, realizes the labor division cooperation mode of gene chip design and gene chip production, and constructs a gene chip industrial chain in a cooperative manner.
Description
Technical Field
The present application relates to a method for preparing a gene chip.
Background
The probes on the traditional gene chip are synthesized once, the probes are placed on the substrate one by utilizing a specific equipment sample application instrument, and the final gene chip is finished through the steps of combining, fixing, cleaning, drying, packaging and the like. The core work of the technology is to fix different probes at different positions of a substrate, and to correspond the positions of the substrate to the probe sequences, so that the fluorescent signals of the positions of the substrate are used for monitoring whether the target sequences exist or not in the final hybridization result detection. In this process, the center distance between two sites on the substrate is generally 450 micrometers, each probe point is only 100-200 micrometers, the sample application amount is in nanoliter level, and the accurate positioning of micrometer level and the tiny sample application amount of nanoliter level are key for manufacturing the gene chip and are needed to be completed by a sample application instrument with high price.
In addition, generally, after the gene chip is manufactured, the probes are fixed on the substrate, and cannot be modified any more, and a user can only perform corresponding target detection by using the probes fixed on the gene chip, so that the gene chip cannot be developed autonomously.
The current gene chip preparation method cannot meet the requirements of users on the diversity and the variability of the detection field.
Disclosure of Invention
The present application provides a method of preparing a gene chip, comprising:
s1, providing a functional substrate with array points formed with probe precursors, wherein the probe precursors comprise anchoring groups, spacer chains and capture areas, the anchoring groups are connected with the capture areas through the spacer chains, the capture areas are a section of DNA sequences, and the probe precursors are connected to the functional substrate through the anchoring groups;
s2, introducing a first template molecule into a first area containing an array point of the probe precursor, wherein the first template molecule comprises a core segment and a universal segment, the core segment and the universal segment are DNA sequences, the sequence of the core segment of the first template molecule is complementary with the sequence of a specific recognition component of a first target probe, and the universal segment of the first template molecule is paired with a capture area of the probe precursor, so that the first template molecule is paired and combined with the capture area of the probe precursor through the universal segment;
s3, adding DNA polymerase and a reaction system thereof into a first region introduced with the first template molecule to perform extension reaction, and extending on the probe precursor to form a specific recognition component of the first target probe to obtain a first complete probe;
s4, increasing the temperature to inactivate the DNA polymerase and dissociating the first template molecule from the first complete probe;
s5, cleaning to obtain the gene chip with the first complete probe single chain.
In one embodiment, the functional substrate is an aldehyde-based substrate and the anchoring group is an amino group.
In one embodiment, the spacer chain is- (CH) 2 ) n-, wherein n is an integer from 4 to 15.
In one embodiment, the capture region is an oligomer of a single first deoxynucleotide;
the universal segment is an oligomer of a single second deoxynucleotide, and the second deoxynucleotide on the universal segment is complementarily paired with the first deoxynucleotide.
In one embodiment, the capture region is an oligomer comprising 15-28 thymidine nucleotides;
the universal segment is an oligomer comprising 15-28 adenine deoxynucleotides.
In one embodiment, the DNA polymerase is selected from the group consisting of e.coli DNA polymerase, T4DNA polymerase, and Klenow enzyme.
In one embodiment, the DNA polymerase is further subjected to a denaturation treatment after inactivation, the denaturation treatment comprising soaking in a solution comprising a denaturing agent.
In one embodiment, the method further comprises:
s2'. Introducing a second template molecule in a second region comprising the array spot of the probe precursor, the second template molecule being different from the first template molecule, the second template molecule comprising a core segment and a universal segment, the sequence of the core segment of the second template molecule being complementary to the sequence of the specific recognition component of the second target probe, and the universal segment of the second template molecule being identical to the universal segment of the first template molecule;
s3', adding DNA polymerase and a reaction system thereof into a second region introduced with the second template molecule to carry out extension reaction, and extending on the probe precursor to form a specific recognition component of the second target probe to obtain a second complete probe.
The present application also relates to a gene chip obtained according to the method of the present application.
The method for preparing the gene chip mainly comprises two stages, wherein the first stage forms a lattice of probe precursors with the same structure on a functional substrate, so that the large-scale production advantage of a chip production enterprise can be exerted, and a gene chip base plate without sequence specificity is manufactured; the second stage is to automatically load probes according to the practical demands of practice on diversified probes, and automatically finish the final production of the gene chips according to the practical demands, so that the advantages of the industrialized batch production of the gene chips can be satisfactorily combined with the diversified detection demands of the users, the development progress of the gene chips can be accelerated, the labor division cooperation mode of the gene chip design and the gene chip production is realized, and the gene chip industrial chain is cooperatively produced.
Drawings
FIGS. 1-4 schematically illustrate the process of the present application;
FIGS. 5-1 to 5-3 schematically illustrate the formation of separate reaction chambers
Fig. 6 shows the detection results of the embodiment.
Detailed Description
The present application is further described in detail below by way of the accompanying drawings and examples. The features and advantages of the present application will become more apparent from the description.
The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Although various aspects of the embodiments are illustrated in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated.
In addition, the technical features described below in the different embodiments of the present application may be combined with each other as long as they do not collide with each other.
The present application provides a method of preparing a gene chip, comprising:
s1, providing a functional substrate with array points formed with probe precursors, wherein the probe precursors comprise anchoring groups, spacer chains and capture areas, the anchoring groups are connected with the capture areas through the spacer chains, the capture areas are a section of DNA sequences, and the probe precursors are connected to the functional substrate through the anchoring groups;
s2, introducing a first template molecule into a first area containing an array point of the probe precursor, wherein the first template molecule comprises a core segment and a universal segment, the core segment and the universal segment are DNA sequences, the sequence of the core segment of the first template molecule is complementary with the sequence of a specific recognition component of a first target probe, and the universal segment of the first template molecule is paired with a capture area of the probe precursor, so that the first template molecule is paired and combined with the capture area of the probe precursor through the universal segment;
s3, adding DNA polymerase and a reaction system thereof into a first region introduced with the first template molecule to perform extension reaction, and extending on the probe precursor to form a specific recognition component of the first target probe to obtain a first complete probe;
s4, increasing the temperature to inactivate the DNA polymerase and dissociating the first template molecule from the first complete probe;
s5, cleaning to obtain the gene chip with the first complete probe single chain.
Probes on a typical gene chip typically comprise 4 components: (1) an anchoring group, (2) a spacer chain, (3) a capture region, and (4) a specific recognition component. The anchoring group is used for being connected with the substrate; the spacer chain can play a role in enhancing hybridization; the capture zone is for pairing with a generic segment of a template molecule for subsequent use so as to bind to the template molecule; the specific recognition component is a specific binding part responsible for recognizing the target nucleic acid in the complete probe structure and is a key region for recognizing the nucleic acid target.
The method for preparing the gene chip by the two-step method comprises two stages, wherein the first stage is the step S1 of the method, the functional substrate with the array points of probe precursors is provided, the probe precursors comprise an anchoring group, a spacer chain and a capture region, the anchoring group is connected with the capture region through the spacer chain, the capture region is a DNA sequence, and the probe precursors are connected to the functional substrate through the anchoring group. In a subsequent process, the capture zone is used to link to a template molecule so that a specific recognition component can be formed extending from the capture zone. The anchor group, spacer and capture region are not specific recognition components for the genetic probe for the identification of the target nucleic acid, and are part of a generic moiety. Thus, the same probe precursor can be used for different gene probes, thereby simplifying the fabrication of the chip.
For the purposes of this application, there are various options for the functional substrate and the anchor groups, as long as the anchor groups are capable of binding to the functional groups on the functional substrate so that the probe precursors can be attached to the functional substrate surface. In one embodiment, the functional substrate is an aldehyde-based substrate and the anchoring group is an amino group from the viewpoint of ease of fabrication and ready availability. The amino anchor groups can readily bind to aldehyde groups on the aldehyde substrate, thereby anchoring the probe precursors to the functional substrate surface. The preparation of aldehyde-based substrates is known in the art and can be described, for example, by reference to chinese patent CN 1325660C, chinese patent CN 101486532B, chinese patent CN 102276863B, chinese patent 202310163369.3, chinese patent 202211682900.X, etc. The aldehyde-based substrates have also been commercially available and are available commercially.
In one embodiment, the spacer chain is- (CH) 2 ) n-, where n is an integer from 4 to 15, for example an integer from 5 to 12. Spacer chain and captureThe regions together allow the final formed complete probe to protrude from the substrate surface, facilitating final gene chip detection.
In the present application, the capture zone used in the method of the present application is a DNA sequence, but it cannot be identical to the sequence of the specific recognition component of the target probe or the coincidence rate with the sequence of the specific recognition component of the target probe is too high to avoid affecting the detection accuracy of the final gene chip. In one embodiment, the capture region is an oligomer of a single first deoxynucleotide for ease of fabrication and to avoid mismatches; for example, an oligomer having 15-28 single first deoxynucleotides. In theory, any single deoxynucleotide oligomer may be used as the capture region, and for example, any of five deoxynucleotides dA (deoxyadenine deoxynucleotide), dG (guanine deoxynucleotide), dC (cytosine deoxynucleotide), dT (thymine deoxynucleotide), dU (uracil deoxynucleotide) may be used to form the capture region. However, the capturing region is a dT oligomer (an oligomer of thymidine nucleotide), particularly an oligomer having 15 to 28 dT, in terms of ease of synthesis, steric hindrance, and the like.
The array of probe precursors can be formed on the functional substrate by various methods known in the art, for example, a spotter can be used to form an array of probe precursor spots of a desired specification on the functional substrate according to a set pitch size and spot size. The anchor group (e.g., amino) of the probe precursor may be bound to a functional substrate (e.g., aldehyde substrate) such that the probe precursor is attached to the functional substrate through the anchor group. Moreover, as the probe precursors are the same, the lattice of the probe precursors can be formed on the functional substrate in one step, the steps of replacing samples, cleaning and the like in the middle of the manufacturing process are not needed, the problems of sample mixing and pollution are avoided, the sample application at all positions can be finished by using one sample application needle, the manufacturing process is very fast, the manufacturing process of a chip is greatly simplified, the production capacity of a chip bottom plate can be greatly improved, and the production cost of the bottom plate is reduced.
The S2-S5 steps of the method belong to the second stage, and a specific recognition component of the target probe is formed by extending from the capture area according to a specific position, so that the complete probe is obtained, and the specific recognition of the complete probe to the target nucleic acid can be realized.
The method comprises S2, introducing a first template molecule into a first area containing an array point of the probe precursor, wherein the first template molecule comprises a core segment and a universal segment, the core segment and the universal segment are DNA sequences, the sequence of the core segment of the first template molecule is complementary with the sequence of a specific recognition component of a first target probe, and the universal segment of the first template molecule is paired with a capture area of the probe precursor, so that the first template molecule is paired and combined with the capture area of the probe precursor through the universal segment.
The template molecule comprises two parts, one part is a universal segment which is a DNA sequence, and the universal segment of the first template molecule is matched with the capture region of the probe precursor, so that the first template molecule is connected with the probe precursor through the universal segment. Thus, the design of the generic section needs to be varied depending on the capture zone of the probe precursor. When the capture region is an oligomer of a single first deoxynucleotide; for example, with an oligomer of 15-28 single first deoxynucleotides, the universal segment is likewise an oligomer of a single second deoxynucleotide, and the second deoxynucleotide on the universal segment is complementarily paired with the first deoxynucleotide. In theory, any single deoxynucleotide oligomer may be used as the capture region, and for example, any of dA (deoxyadenine deoxynucleotide), dG (guanine deoxynucleotide), dC (cytosine deoxynucleotide), dT (thymine deoxynucleotide), dU (uracil deoxynucleotide) may be used to form the universal segment, provided that the second deoxynucleotide of the universal segment is capable of pairing with the first deoxynucleotide of the capture region. For example, when the single deoxynucleotide forming the capture region is dA, the single deoxynucleotide forming the universal segment may be dT or dU; when the single deoxynucleotide forming the capture region is dG, the single deoxynucleotide forming the universal segment may be dC; when the single deoxynucleotide forming the capture region is dT, the single deoxynucleotide forming the universal segment may be dA; when the single deoxynucleotide forming the capture region is dC, the single deoxynucleotide forming the universal segment may be dG. However, as described above, when the capturing region is dT oligomer (oligomer of thymidine nucleotide), particularly oligomer having 15 to 28 dT, the universal segment is dA oligomer (oligomer of adenine deoxynucleotide), particularly oligomer having 15 to 28 dA.
The other part of the template molecule is a core segment which is also a DNA sequence, and the sequence of the core segment is complementary with the sequence of the specific recognition component of the first target probe, so that the specific recognition component of the target probe is formed by extending on the probe precursor according to the sequence of the core segment in the subsequent extension reaction. The sequences of the core segments are designed autonomously by the user according to the specific requirements of the user, target sequences to be detected and the like, so that the user can independently develop the gene chip, and the requirements of diversity and variability of the user are met.
In one embodiment, different template molecules, which may have the same universal segment but different core segment sequences, may be introduced in different areas of the array spot where the probe precursor is formed on the functional substrate, thereby forming different target probe areas on one substrate, enabling multipurpose detection. The process can be designed independently by the user according to the own requirements, and the requirements of diversity and variability of the user are further realized.
Thus, in one embodiment, the method further comprises:
s2'. Introducing a second template molecule in a second region comprising the array spot of the probe precursor, the second template molecule being different from the first template molecule, the second template molecule comprising a core segment and a universal segment, the sequence of the core segment of the second template molecule being complementary to the sequence of the specific recognition component of the second target probe, and the universal segment of the second template molecule being identical to the universal segment of the first template molecule;
s3', adding DNA polymerase and a reaction system thereof into a second region introduced with the second template molecule to carry out extension reaction, and extending on the probe precursor to form a specific recognition component of the second target probe to obtain a second complete probe.
To avoid interference in the various regions, it is often necessary to space the different regions apart. For example, the method disclosed in CN1434296a may be used to form a separate reaction isolation region such that the extension reactions and the like that follow in the separate reaction isolation region do not interfere with each other. Alternatively, a closed reaction chamber may be formed over the chip by a PDMS (polydimethylsiloxane) material, and a desired material, such as a template molecule, a DNA polymerase, a reactant, etc., may be introduced into the reaction chamber by puncturing the flexible PDMS material with a loading gun.
After introduction of the template molecule, the temperature is adjusted such that the capture zone is paired with the universal segment, thereby allowing the template molecule to be linked to the probe precursor through the universal segment.
Then, DNA polymerase and a reaction system thereof are added to carry out an extension reaction. And introducing DNA polymerase and a reaction system (comprising reactants, reaction temperature and the like) into the region into which the template molecule is introduced to perform extension reaction, and forming a specific recognition component of the target probe on the probe precursor by taking a core segment of the template molecule as a primer. The extension reaction process is similar to the PCR reaction, and the DNA polymerase used may be a DNA polymerase used in the PCR reaction such as E.coli DNA polymerase, T4DNA polymerase or Klenow enzyme, and the reactant is a solution containing various nucleotide molecules commonly used in the PCR reaction. However, since the extension reaction does not require multiple extensions, only one extension reaction is required, and all probes in the reaction region can be formed in one extension reaction, the use of a high temperature-resistant DNA polymerase is not required, the reaction requirement is low, the operation is easy, and the cost can be reduced. A preferred DNA polymerase may be the klenow enzyme, which is inactivated at 70℃for 20 minutes. The extension reaction may be carried out at room temperature, for example for more than 5 minutes, to promote mixing and adequate reaction.
In the process, the extension reaction based on DNA polymerase is carried out once, a temperature changing system of the traditional PCR reaction is not needed in the reaction process, and the reaction time is finished in the order of minutes. Secondly, the combination process of the template molecules and the probe precursors on the functional substrate and the extension reaction in the presence of DNA polymerase can be carried out separately, the template molecules are added to complete the annealing combination of the probe precursors and the template molecules, and then the DNA polymerase system is added to carry out extension.
To avoid contamination, it is also generally necessary to inactivate the DNA polymerase and dissociate the first template molecule from the first complete probe after the extension reaction. For example, the DNA polymerase may be inactivated by incubation in an oven at 75 ℃ for a period of time.
After the inactivation of the DNA polymerase, a denaturation treatment is also performed, which comprises soaking in a solution comprising a denaturing agent, for example, soaking in a solution comprising SDS.
After the extension reaction, the template molecule is attached to the probe by base-complementary pairing only and is directly attached to the substrate. After dissociation of the first template molecule from the first complete probe, the template molecule can be removed by washing, so that only the probe portion remains on the substrate, but no template molecule portion, thereby obtaining the final gene chip with complete probe single strands.
Fig. 1-4 schematically illustrate the process of the present application, which is further described below in connection with fig. 1-4.
In a first stage, a plurality of array spots of probe precursors are formed on sites 2 of functional substrate 1, the probe precursors on the array spots comprising anchor groups 5, spacer chains (not shown) and capture zones 6, the anchor groups 5 being attached to the capture zones 6 by the spacer chains (not shown), and the anchor groups 5 being attached to the functional substrate 1. This step can be achieved industrially by making the base plate of the gene chip, and attaching the same capture area 6 at a specific site.
To facilitate the subsequent steps, PDMS materials were used to form individual reaction chambers in certain areas of the spots. Positioning frames 14 with positioning holes 15 are placed on the functional substrates 1 after sample application, as shown in fig. 5-1; then, a PDMS adhesive film 12 (as shown in fig. 5-2) with a plurality of reaction chambers 13 with one end closed is sleeved on the functional substrate 1, so that each reaction chamber 13 is in sealing contact with the bottom end of the substrate to form a separate reaction chamber together for performing the subsequent steps. Fig. 5-3 show schematic diagrams of the arrangement of the PDMS adhesive film 12 in a partial area on the functional substrate 1, in which 12A is the sealing area where the PDMS adhesive film contacts the substrate, 13A is the sealing junction of the reaction chamber and the substrate, and 17 is the spot.
The second stage comprises the following main steps:
2.1 step, adding a template molecule to the reaction chamber to introduce the template molecule at site 2, wherein the template molecule comprises a general segment 7-1 and a core segment 7-2. Since the universal segment 7-1 is paired with the capture zone 6, the template molecule is linked to the probe precursor, but not directly to the functional substrate 1, as in FIG. 2.
2.2, adding DNA polymerase into the reaction chamber and performing one-time extension reaction under corresponding reaction conditions (reactants and temperature), thereby forming a specific recognition component 8 of the target probe by extension on the capture zone 6 by taking the core segment 7-2 as a template, and forming a local nucleic acid double strand, as shown in FIG. 3.
2.3, inactivating the DNA polymerase by raising the temperature, dissociating the first template molecule from the first complete probe, and performing a washing treatment to keep only single strands containing the target probes 9, thereby forming complete target probes 9 on the functional substrate 1, as shown in FIG. 4.
The whole second stage can be realized by a user according to actual needs, the specific recognition component of the probe is automatically extended on a specific site, and finally the specific probe required by user detection is formed and the whole manufacturing process of the gene chip is completed.
Examples:
the first stage: preparation of Gene chip baseboard
(1) Glass surface activation: cleaning the surface of a low-fluorescence white glass sheet to remove oil stains and dust, performing surface activation by using concentrated sulfuric acid and hydrogen peroxide (95:5 volume ratio), and performing surface amino modification after washing;
(2) Amino modification: amino modifier is added to carry out amino modification: using 1mol/L acetone solution of 3-aminopropyl triethoxysilane, reacting for 0.5-2 hours at room temperature, and assisting in slight oscillation to obtain an amino modified substrate;
(3) Aldehyde group modification: dialdehyde compound modification is carried out on the amino modified substrate: using 1mol/L acetone solution of terephthalaldehyde, and reacting for 0.5-2 hours at room temperature to obtain an aldehyde group substrate;
(4) The oligo dT with amino modification and connection with alkane chain is synthesized (synthesis of Shanghai, kunspanol and company are synthesized, 15-22 dT oligomers, alkane carbon number is 6-12), and the dot diameter can be controlled in the range of 100-250 micrometers by piezoelectric spraying dot onto the substrate by using a dot sample applicator (inlet device: GESIM Nano Plotter NP 2.1.1/E).
Size parameters: the Boottan glass substrate has the specification that: 75mm long, 25mm wide and 1mm thick.
A gene chip floor was obtained, with the same oligo dT at all sites. The probe center distance of the gene chip is preferably 450 micrometers, wherein the diameter of the probe spot is 200 micrometers. Every four probe spots are in a group, and correspond to one site on the gene chip, and the distance between the nearest two probes between the sites is 1350 micrometers (probe spotting is performed at intervals of 2 rows or 2 columns).
Thereafter, a PDMS film was placed on the spot areas as shown in FIGS. 5-1 to 5-3, forming separate reaction chambers each comprising 4 spots.
And a second stage: full-length probes were prepared using DNA polymerase (second stage of two-step preparation of gene chip).
The full length probe of this embodiment comprises 4 components: an anchor group (amino group), a spacer chain (alkane chain), a capture region (oligo dT) and a specific recognition module; the connection sequence is as follows: amino-alkane chain-oligo dT-specific recognition module (extension formation). The 4 th component specific recognition component can specifically recognize target nucleic acid, is a key of gene chip recognition, and other parts can be simply called universal components. The universal component of the probe is attached to the substrate in the first step of fabricating the base plate, and the specific recognition component is formed by DNase catalysis using oligo dT as a primer in the second step.
(1) The synthesis of DNA templates is delegated.
The DNA template with 3' end oligo dA modification is entrusted to be synthesized, the 5' end is the core segment of the template, the template is the specific recognition component part of the synthesized probe, and the 3' end oligo dA is used for pairing and combining with the oligodeoxythymidine in the probe universal component on the gene chip base plate. Different templates correspond to different sites of the gene chip, so that the full-length probe can identify different target nucleic acids. (entrusted Shanghai engineering Synthesis, kunshanpu and Co., ltd., template for the production of a Probe specific recognition Assembly, 15-22 dA oligomers in 3' with no need for alkane Strand modification.)
The specific sequence is as follows:
(2) The DNA template molecules were formulated to appropriate concentrations using hybridization solution for binding to oligo dT.
(3) Template molecules are respectively injected into the independent reaction chambers according to the plan by using a disposable fine gun head, and the sample loading amount of each reaction chamber is 0.8 microliter.
(4) Adjusting the temperature of the reaction chamber to enable oligo dT of a probe universal component on a base plate of the gene chip to be paired with oligo dA on a template molecule;
(5) Adding 0.5 microliter of klenow enzyme (bio, cat No. B110065) and its reaction system (containing four deoxynucleotides) at 37 ℃ for more than 5 minutes (to promote mixing and complete reaction);
(6) Incubate in an oven at 75deg.C for 30 minutes (inactivate klenow enzyme);
(7) And removing the PDMS film on the substrate, soaking in a buffer solution for 10 minutes, washing with a washing solution containing SDS, washing with water, and drying for later use. And (5) finishing the manufacturing process of the gene chip.
Result detection
And (3) chip detection: the hybridization detection method is the same as that described in patent ZL 2021115939968, an encapsulated gene chip. And packaging the gene chip obtained in the three steps, and hybridizing and cleaning according to a method disclosed in patent ZL 2021115939968 to finish the hybridization process of the fluorescent marked target DNA and the gene chip.
The packaged gene chip after hybridization reaction is put into a gene chip detector, the wavelength of an excitation light source is 630nm, detection is carried out by using the emission wavelength of 670nm, an image of a detection result is obtained by a CMOS camera, and analysis of the chip hybridization result is carried out by using matched analysis software. The detection results are shown in FIG. 6.
In FIG. 6, the left side is a probe region prepared by a two-step method, and each hybridization point is round, full and clear in boundary, because the region is a complete probe structure, the probe is provided with a specific recognition component and can be combined with a fluorescent-labeled target nucleic acid; the right side only has general components, can not recognize and combine target nucleic acid, and has no fluorescence.
The present application has been described in connection with the preferred embodiments, but these embodiments are merely exemplary and serve only as illustrations. On the basis of this, many alternatives and improvements can be made to the present application, which fall within the scope of protection of the present application.
Claims (9)
1. A method of preparing a gene chip comprising:
s1, providing a functional substrate with array points formed with probe precursors, wherein the probe precursors comprise anchoring groups, spacer chains and capture areas, the anchoring groups are connected with the capture areas through the spacer chains, the capture areas are a section of DNA sequences, and the probe precursors are connected to the functional substrate through the anchoring groups;
s2, introducing a first template molecule into a first area containing an array point of the probe precursor, wherein the first template molecule comprises a core segment and a universal segment, the core segment and the universal segment are DNA sequences, the sequence of the core segment of the first template molecule is complementary with the sequence of a specific recognition component of a first target probe, and the universal segment of the first template molecule is paired with a capture area of the probe precursor, so that the first template molecule is paired and combined with the capture area of the probe precursor through the universal segment;
s3, adding DNA polymerase and a reaction system thereof into a first region introduced with the first template molecule to perform extension reaction, and extending on the probe precursor to form a specific recognition component of the first target probe to obtain a first complete probe;
s4, increasing the temperature to inactivate the DNA polymerase and dissociating the first template molecule from the first complete probe;
s5, cleaning to obtain the gene chip with the first complete probe single chain.
2. The method of claim 1, wherein the functional substrate is an aldehyde substrate and the anchoring group is an amino group.
3. The method of claim 1, wherein the spacer chain is- (CH) 2 ) n-, wherein n is an integer from 4 to 15.
4. The method of claim 1, wherein the capture region is an oligomer of a single first deoxynucleotide;
the universal segment is an oligomer of a single second deoxynucleotide, and the second deoxynucleotide on the universal segment is complementarily paired with the first deoxynucleotide.
5. The method of claim 4, wherein the capture region is an oligomer comprising 15-28 thymidine nucleotides;
the universal segment is an oligomer comprising 15-28 adenine deoxynucleotides.
6. The method of claim 4, wherein the DNA polymerase is selected from the group consisting of escherichia coli DNA polymerase, T4DNA polymerase, and Klenow enzyme.
7. The method of claim 1, wherein after inactivating the DNA polymerase, a denaturation treatment is further performed, the denaturation treatment comprising soaking in a solution comprising a denaturant.
8. The method of any of claims 1-7, wherein the method further comprises:
s2'. Introducing a second template molecule in a second region comprising the array spot of the probe precursor, the second template molecule being different from the first template molecule, the second template molecule comprising a core segment and a universal segment, the sequence of the core segment of the second template molecule being complementary to the sequence of the specific recognition component of the second target probe, and the universal segment of the second template molecule being identical to the universal segment of the first template molecule;
s3', adding DNA polymerase and a reaction system thereof into a second region introduced with the second template molecule to carry out extension reaction, and extending on the probe precursor to form a specific recognition component of the second target probe to obtain a second complete probe.
9. A gene chip obtainable by a method according to any one of claims 1-8.
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