CN114350774A - Detection method for chip surface solid phase primer shearing efficiency - Google Patents
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Abstract
The invention discloses a method for detecting the shearing efficiency of a chip surface solid phase primer, which comprises the following steps: (1) hybridizing a template complementary to the primer immobilized on the chip to the chip; the primer contains a site which can be cut; the template extends n nt from the 3' end of the primer; (2) performing fluorescence generation sequencing on the surface of the chip, and extending only one base on each primer to obtain average fluorescence increment int 1; (3) treating the chip with a shearing liquid, and unwinding; (4) hybridizing the chip with the template again, performing fluorescence generation sequencing, and adding a substrate containing A, C, G, T four bases to obtain average fluorescence increment int 2; (5) and calculating to obtain the shearing efficiency. The invention adopts a method of performing fluorescence sequencing on the surface of the chip, and achieves the aim of detecting the shearing efficiency of the solid phase primer on the surface of the chip in situ. The method can be popularized and applied to the determination of the shearing efficiency of the primers on various solid-phase surfaces.
Description
Technical Field
The invention relates to a method for detecting the shearing efficiency of a chip surface solid phase primer, belonging to the technical field of gene sequencing.
Background
The chip solid phase primer shearing is a common step when amplification is carried out on a chip, and various amplification technologies represented by bridge amplification of Illumina company can use the reaction, and the reaction mainly has the function of shearing double-stranded DNA amplified on the solid phase surface into single strands for subsequent hybridization primers to carry out sequencing reaction. If the shearing step is not carried out, the solid phase double-stranded DNA can not normally hybridize with a sequencing primer to carry out a sequencing reaction, so that the step is an extremely important step in an amplification link. At present, the validation of the shearing efficiency is basically carried out by validating the enzyme activity in a liquid phase alone or combining with the downstream sequencing quality; or hybridization fluorescent probe to detect the density of the solid phase primer, but the detection precision of the method is very low. In the prior art, a method for accurately and directly detecting the shearing efficiency of the solid phase primer on the surface of the chip is not provided.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for detecting the shearing efficiency of a solid phase primer on the surface of a chip, which is used for solving the problem that the shearing efficiency of the primer cannot be directly and accurately detected on the chip in various solid phase amplification processes.
The invention is realized by the following technical scheme:
a detection method for the shearing efficiency of a chip surface solid phase primer comprises the following steps:
(1) hybridizing a template complementary to the primer immobilized on the chip to the chip; the primer contains a site which can be cut; the template extends n nt from the 3' end of the primer for primer extension, wherein n is an integer (preferably 10-20) greater than or equal to 2;
further, the cleavable site is a specifically modified base such as: dU base, 8-oxodeoxyguanosine (8-oxo-dG), Tetrahydrofuran (THF) -modified base;
further, the length of the primer is 20-45 nt;
(2) performing fluorescence generation sequencing on the surface of the chip, and extending only one base on each primer to obtain average fluorescence appreciation int1, namely a unit signal when extending one base on each primer;
furthermore, the specific manner of extending only one base on each primer is as follows: during sequencing reaction, only one base is contained in the added substrate, and the base is the first base extended by the primer;
(3) treating the chip with a shearing liquid, and unwinding;
after the primers on the chip are successfully cut, the primers can be separated from the solid phase through unwinding, and the upper template can not be hybridized, so that the primers can not be extended continuously in the subsequent sequencing reaction process and a sequencing signal can not be obtained;
in the shearing process, the shearing liquid acts on the base position of the special modification, and the primer is sheared into two sections at the base position; after the derotation, the solid phase primer is broken from the shearing part and derotated into a liquid phase, so that the amplification product is recovered into a single chain and can be smoothly combined with a sequencing primer;
the selection of the shear fluid is related to the specially modified base on the primer; such as: if the specially modified base on the primer is dU base, selecting USER enzyme mixed liquor (mainly comprising uracil DNA glycosylase and endonuclease VIII) as a shearing liquid; selecting an enzyme solution containing Fpg as a cleavage solution if the specifically modified base on the primer is 8-oxodeoxyguanosine; if the specially modified base on the primer is a base modified by tetrahydrofuran, selecting an enzyme solution containing Endonuclease IV (Nfo) as a shearing solution; for some specially modified solid phase primers, reagents such as sodium periodate and the like can be selected as a shearing fluid;
(4) hybridizing the chip subjected to the shearing reaction with the template again, performing fluorescence sequencing, wherein the added substrate contains A, C, G, T four bases, so that the uncut primer can completely extend the rest n-1 bases to obtain the average fluorescence increment int 2;
(5) the shear efficiency is calculated by the following formula:
further, the surface of the chip is fixedly loaded with microspheres, and the microspheres are connected with primers; furthermore, the surface of the microsphere is connected with two amplification primers in advance, and at least one amplification primer contains a site which can be cut; still further, biotin is arranged on the microspheres as a group for immobilization, modified streptavidin is arranged on the chip, and the microspheres are connected to the chip through a specific reaction of the biotin and the streptavidin; still further, the diameter of the microsphere is 0.3-5 microns, preferably 0.5-4 microns, and more preferably 1-2 microns.
The at least one amplification primer contains a cleavable site, which means that only one amplification primer of the two amplification primers contains a cleavable site, or both amplification primers contain 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 cleavage conditions 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.
The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip adopts a method of performing fluorescence generation sequencing on the surface of the chip, and achieves the aim of detecting the shearing efficiency of the solid phase primer on the surface of the chip in situ. Compared with indirect modes such as detecting the enzymatic activity of the shearing liquid or sequencing effect in a liquid phase, the method provided by the invention is more direct, and the shearing efficiency of the solid phase primer on the surface of the chip is directly detected. Meanwhile, the method can achieve higher detection precision, and the length of the template can be adjusted to realize amplification or reduction of the reaction signal so as to better adjust the detection range of the method. Moreover, the method has wide application range, and can be used for detecting the shearing efficiency of primers on various solid phase surfaces (such as glass dish or microsphere surfaces).
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Drawings
FIG. 1: the principle schematic diagram of the detection method of the chip surface solid phase primer shearing efficiency is shown in the invention.
FIG. 2: fluorescence results of the same site in example 3 (before cleavage).
FIG. 3: fluorescence results of the same site in example 3 (after cleavage).
Detailed Description
The present invention will be further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and that no limitation of the invention is intended. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The chip of the invention includes but is not limited to gene sequencing chip. The gene sequencing chip is a general chip having a fluid inlet and outlet and a reaction chamber. Similar gene sequencing chips, such as CN201710574174.2, cn201710630287.x, have been disclosed many times in applicant's prior invention patent applications, the contents of which can be incorporated by reference into the present invention as necessary.
In the invention, "shearing" is a common step of molecular biology and surface amplification, and the common shearing refers to shearing a base and a chemical site 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; other cleavage methods may be used as alternatives to restriction enzymes, including chemical cleavage (e.g., cleavage of a diol bond with periodate), or cleavage of an abasic site by exposure to heat or base, photochemical cleavage, and the like. The site of cleavage is a specifically modified base such as: dU bases, 8-oxodeoxyguanosine (8-oxo-dG), Tetrahydrofuran (THF) modified bases, and may also include deoxyhypoxanthine, apurinic/apyrimidinic sites, dihydroxyl sites, and the like.
The solid phase primer of the present invention is a conventional description in the art, and refers to a primer bound to the surface of a solid phase carrier, which is distinguished from a liquid phase free primer, and the solid phase carrier includes a chip surface or a carrier such as a microsphere.
The method is characterized in that n nt extends from the 3' end of a primer of a template nucleic acid, namely n nucleotide molecules in the template nucleic acid are not subjected to base complementary pairing with the primer when the primer is hybridized with the template, and then at most n nucleotide molecules can be extended, wherein the nt is nucleotide.
The fluorescence generation sequencing of the invention refers to that the fluorescence generation nucleotide polymerase reaction is utilized, and the fluorescence change (light intensity and spectrum) of a fluorescence generation fluorophore is detected, so that the information of the polymerase reaction can be obtained. The fluorogenic nucleotide polymerase reaction uses fluorogenic nucleotides, nucleic acid polymerase (DNA polymerase), phosphatase, together with a nucleic acid template. Firstly, the DNA polymerase polymerizes the fluorescence generating nucleotide into the nucleic acid template to release phosphorylated fluorescence generating fluorophore, and then the phosphorylation is further hydrolyzed by phosphatase to remove the phosphate to release the fluorescence generating fluorophore with changed fluorescence state.
Example 1 detection of cleavage efficiency of solid phase primer on chip surface
The steps are as follows (the principle is shown in figure 1):
(1) a chip to be tested for shear reaction efficiency was prepared. In this example, a chip provided by a serna organism is used, and the chip has millions of microwells, and microspheres with modified primers are immobilized in the microwells; the chip can be used for library amplification and fluorescence generation high-throughput sequencing reaction. The sequences of the primers are shown below (containing dU bases, being cleavable sites):
5’-dUGAAGGTGTGCCATCTCATCCCTGCGTGTCTCCGACTCAG-3’。
(2) template hybridization: the sequencing template was diluted to 2 μ M with 5x SSC buffer and then added to the chip. Placing the chip on a flat PCR instrument for template hybridization, wherein the hybridization process comprises the following steps: 96 ℃ for 30 s; 0.05 ℃/s; 10s at 40 ℃; 25 ℃ and forever. After the hybridization process, the chip was removed and washed with fluorogenic sequencing buffer.
The sequence of the template is as follows:
5’-TGGCTTCGGAGACAACTGAGTCGGAGACACGCAGGGATGAGATGGCACACCTTCAAT-3’。
(3) preparing a sequencing reaction solution before shearing reaction: from the sequences of the primers and the template, the first base to be extended by the primers is a T base, and therefore, a fluorogenic sequencing reaction solution containing only the T base is prepared: 12. mu.L of fluorophore-modified dT base (initial concentration 20mM, final concentration 40. mu.M), 321.6. mu.L of fluorogenic sequencing enzyme mixture, 5666.4. mu.L of fluorogenic sequencing buffer, and 6000. mu.L of total volume.
(4) Sequencing reaction before cleavage reaction: and (3) placing the cleaned chip on a BOOTES sequencer of the Saina creature, and then placing the sequencing reaction solution prepared in the step (3) at a position corresponding to the sequencer. The sequencing script of the sequencer is selected and run, and the sequencer enters the corresponding reagent to perform an extension reaction and record the fluorescence value int 1.
(5) And (3) shearing reaction: the shear reaction fluid is added to the chip. Then the chip is placed on a flat PCR instrument or a sequencer for reaction, and the reaction is carried out for 30min at 37 ℃. After the reaction, the chip was removed and washed with fluorogenic sequencing buffer.
The shearing reaction liquid is a USER enzyme mixed liquid: USER enzyme 4. mu.L, CutSmart buffer 40. mu.L, water 356. mu.L, total volume 400. mu.L.
(6) Unwinding: after the shearing reaction is finished, taking down the chip, washing the chip by using 1mL of fluorescence generation sequencing buffer solution, adding 1mL of formamide for reacting at room temperature for 3min for derotation, and washing the chip by using 1mL of fluorescence generation sequencing buffer solution again.
(7) Template hybridization: the sequencing template was diluted to 2 μ M with 5x SSC buffer and then added to the chip. Placing the chip on a flat PCR instrument for template hybridization, wherein the hybridization process comprises the following steps: 96 ℃ for 30 s; -0.05 ℃/s; 10s at 40 ℃; 25 ℃ and forever. After the hybridization process, the chip was removed and washed with fluorogenic sequencing buffer.
(8) Preparing a sequencing reaction solution after a shearing reaction, wherein the sequencing reaction solution comprises the following components: 12 μ L of fluorophore-modified dA base (initial concentration 20mM, final concentration 40 μ M), 12 μ L of fluorophore-modified dT base (initial concentration 20mM, final concentration 40 μ M), 12 μ L of fluorophore-modified dC base (initial concentration 20mM, final concentration 40 μ M), 12 μ L of fluorophore-modified dG base (initial concentration 20mM, final concentration 40 μ M), 321.6 μ L of fluorogenic sequencing enzyme mixture, 5630.4 μ L of fluorogenic sequencing buffer, and a total volume of 6000 μ L.
(9) Sequencing reaction after cleavage reaction: and (3) placing the cleaned chip on a BOOTES sequencer of the Saina creature, and then placing the sequencing reaction solution prepared in the step (8) at a position corresponding to the sequencer. The sequencing script of the sequencer was selected and run, and the sequencer entered the corresponding reagents for the extension reaction and recorded the fluorescence value int 2.
(10) Calculating the cleavage efficiency according to the following formula, wherein n is the number of total extendible bases at the 3' end of the solid phase primer (n-15); as a result: the shear efficiency was 98.72%.
Example 2 detection of cleavage efficiency of solid phase primer on chip surface
The components of the shearing fluid are as follows: 2.8. mu.L of USER enzyme, 40. mu.L of CutSmart buffer, 357.2. mu.L of water, and 400. mu.L of total volume. The rest is the same as example 1.
As a result: the shear efficiency was 97.64%.
Example 3 detection of cleavage efficiency of solid phase primer on chip surface
The components of the shearing fluid are as follows: 20. mu.L of USER enzyme, 40. mu.L of CutSmart buffer, 160. mu.L of glycerol, 180. mu.L of water, and a total volume of 400. mu.L. The rest is the same as example 1. As a result: the shear efficiency was 99.77%.
The fluorescence results for the same position are shown in FIG. 2 (before the shear reaction) and FIG. 3 (after the shear reaction), where the fluorescence intensity has been adjusted to the same contrast.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the present disclosure. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (10)
1. A detection method for the shearing efficiency of a solid phase primer on the surface of a chip is characterized by comprising the following steps:
(1) hybridizing a template complementary to the primer immobilized on the chip to the chip; the primer contains a site which can be cut; the template extends n nt from the 3' end of the primer for primer extension, wherein n is an integer greater than or equal to 2;
(2) performing fluorescence generation sequencing on the surface of the chip, and extending only one base on each primer to obtain average fluorescence increment int 1;
(3) treating the chip with a shearing liquid, and unwinding;
(4) hybridizing the chip subjected to the shearing reaction with the template again, performing fluorescence sequencing, and adding a substrate containing A, C, G, T four bases to obtain average fluorescence increment int 2;
(5) the shear efficiency is calculated by the following formula:
2. the method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 1, which is characterized in that: the cleavable site is a specifically modified base.
3. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 2, wherein the method comprises the following steps: the specially modified base is selected from dU base, 8-oxo deoxyguanosine and tetrahydrofuran modified base.
4. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 3, wherein the method comprises the following steps: if the specially modified base is dU base, selecting the USER enzyme mixed solution as a shearing solution; selecting an enzyme solution containing Fpg as a shearing solution if the specifically modified base is 8-oxodeoxyguanosine; if the specifically modified base is a tetrahydrofuran modified base, an enzyme solution containing Endonuclease IV (Nfo) is selected as a shearing solution.
5. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 4, wherein the method comprises the following steps: the shearing liquid of the USER enzyme mixed liquid comprises the following components:
4 mu L of USER enzyme, 40 mu L of CutSmart buffer solution and 356 mu L of water, wherein the total volume is 400 mu L;
or: 2.8 mu L of USER enzyme, 40 mu L of CutSmart buffer solution and 357.2 mu L of water, wherein the total volume is 400 mu L;
or: 20. mu.L of USER enzyme, 40. mu.L of CutSmart buffer, 160. mu.L of glycerol, 180. mu.L of water, and a total volume of 400. mu.L.
6. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 1, which is characterized in that: the length of the primer is 20-45 nt.
7. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 1, which is characterized in that: n is an integer of 2 or more, preferably 10 to 20.
8. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 1, which is characterized in that: the specific way of extending each primer by only one base is as follows: when sequencing reaction is carried out, only one base is contained in the added substrate, and the base is the first base extended by the primer.
9. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 1, which is characterized in that: the surface of the chip is fixedly loaded with microspheres, and the microspheres are connected with primers.
10. The method for detecting the shearing efficiency of the solid phase primer on the surface of the chip according to claim 9, wherein the method comprises the following steps: the surface of the microsphere is connected with two amplification primers in advance, and at least one amplification primer contains a site which can be cut.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0324456D0 (en) * | 2003-10-20 | 2003-11-19 | Isis Innovation | Parallel DNA sequencing methods |
| WO2008104791A1 (en) * | 2007-03-01 | 2008-09-04 | Oxitec Limited | Methods for detecting nucleic acid sequences |
| CN102643893A (en) * | 2011-02-16 | 2012-08-22 | 陈国燕 | DNA sequencing method based on primer array chip |
| CN103388024A (en) * | 2013-07-04 | 2013-11-13 | 徐州医学院 | Bridge-PCR-based method for detecting DNA hydroxymethylation |
-
2022
- 2022-01-28 CN CN202210104006.8A patent/CN114350774A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0324456D0 (en) * | 2003-10-20 | 2003-11-19 | Isis Innovation | Parallel DNA sequencing methods |
| WO2008104791A1 (en) * | 2007-03-01 | 2008-09-04 | Oxitec Limited | Methods for detecting nucleic acid sequences |
| EP2129802B1 (en) * | 2007-03-01 | 2016-04-13 | 360 Genomics Limited | Methods for detecting nucleic acid sequences |
| CN102643893A (en) * | 2011-02-16 | 2012-08-22 | 陈国燕 | DNA sequencing method based on primer array chip |
| CN103388024A (en) * | 2013-07-04 | 2013-11-13 | 徐州医学院 | Bridge-PCR-based method for detecting DNA hydroxymethylation |
Non-Patent Citations (3)
| Title |
|---|
| 张小燕, 左明雪, 张占军, 王忠, 李瑶: "用基因芯片检测单核苷酸多态性反应原理", 中国生物工程杂志, no. 11, pages 57 - 61 * |
| 张明哲;张晓峰;陈笑梅;陈曦;吴姗;陈吴健;: "1种高特异性的酶连接探针杂交芯片及其在转基因检测中的应用", 中国食品学报, no. 10, pages 228 - 232 * |
| 文思远, 张敏丽, 陈苏红, 王升启: "基因芯片结合可环化探针对单碱基突变的检测", 军事医学科学院院刊, no. 06, pages 33 - 36 * |
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