CN112899346A - Nucleotide for reducing PCR (polymerase chain reaction) nonspecific amplification and design method and application thereof - Google Patents
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- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 102
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- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 39
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- 238000003752 polymerase chain reaction Methods 0.000 title abstract description 74
- 238000012408 PCR amplification Methods 0.000 claims abstract description 20
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- 239000007983 Tris buffer Substances 0.000 claims description 7
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- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 7
- -1 pyridone azo compound Chemical class 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 7
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
The invention provides a nucleotide for reducing PCR non-specific amplification and a design method and application thereof, wherein the nucleotide for reducing PCR non-specific amplification comprises a sequence A and a sequence B, the sequence A comprises at least 6 nucleotides, and the nucleotide in the sequence A is formed by selecting three bases from A, T, C and G; the sequence B comprises at least 4 nucleotides, the nucleotides of the sequence B can be any base, and the sequence B can form a three-dimensional structure. The invention provides a nucleotide for reducing PCR non-specific amplification and a design method and application thereof, which can complete polymerase chain reaction, detect a target gene sequence and obviously control the non-specific amplification in the PCR amplification process.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to nucleotide for reducing PCR (polymerase chain reaction) nonspecific amplification and a design method and application thereof.
Background
The Polymerase Chain Reaction (PCR) technique is one of the core techniques in molecular biology. From the invention, about 30 years to date, a new technology and a new application developed based on the PCR technology emerge endlessly, and the development of the life science field is promoted. However, the generation of non-specific products in PCR amplification greatly restricts the detection information flux of the PCR technology and also restricts the further application and development of the PCR technology. Non-specific products are typically generated as a result of non-specific amplification within the PCR system due to primer-to-primer, and primer-to-non-target template binding. The generation of non-specific products consumes the substrate in the system, reduces the strength of specific signals and simultaneously reduces the signal-to-noise ratio of detection results.
Therefore, there is a need to develop a PCR amplification primer or probe to reduce or eliminate the non-specific amplification problem during PCR amplification.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a nucleotide for reducing PCR non-specific amplification and a design method and application thereof, which can complete polymerase chain reaction, detect a target gene sequence and obviously control the non-specific amplification in the PCR amplification process, such as reducing primer dimer and the like.
The first aspect of the invention provides a design method for reducing the nucleotide amplified non-specifically by PCR, the nucleotide amplified non-specifically by PCR comprises a sequence A and a sequence B, the sequence A comprises at least 6 nucleotides, and the nucleotide in the sequence A selects three bases from A, T, C and G as the constitution; the sequence B comprises at least 4 nucleotides, the nucleotides of the sequence B can be any base, and the sequence B can form a three-dimensional structure. By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and the nonspecific amplification existing in the PCR amplification process is obviously controlled.
Preferably, for the design method as described above, further comprising: pretreating the nucleotide for reducing the PCR non-specific amplification, wherein the pretreatment method comprises the following steps: connecting the sequence A with the sequence B to obtain combined nucleotide; adding the combined nucleotide into a buffer solution, heating to 65-85 ℃, preserving heat for 5-10 minutes, then cooling to 0-40 ℃, and preserving heat for 20-30 minutes. By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and nonspecific amplification in the PCR amplification process is obviously controlled, such as reduction of primer dimer and the like.
Preferably, for the design method as described above, further comprising: the buffer solution comprises the following components: 300+ -10 mM NaCl, 5 + -1 mM MgCl 220. + -. 5 mM Tris (pH 7.6). By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and nonspecific amplification in the PCR amplification process is obviously controlled, such as reduction of primer dimer and the like.
Preferably, for the design method as described above, further comprising: the buffer solution comprises 0.001-0.003 mu M of pyridone azo compound. By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and nonspecific amplification in the PCR amplification process is obviously controlled, such as primer dimer reduction and the like; the primers can be promoted to rapidly enter the stable state of the polymerase chain reaction.
The second aspect of the present invention provides a nucleotide prepared according to the design method.
In a third aspect, the invention provides a use of a nucleotide according to claim 5 in PCR amplification.
Preferably, for the application as described above, further comprising: in the PCR amplification, DNA polymerase and 0.001-0.003 mu M pyridone azo compound are used. By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and nonspecific amplification in the PCR amplification process is obviously controlled, such as primer dimer reduction and the like; the primers can be promoted to rapidly enter the stable state of the polymerase chain reaction.
The fourth aspect of the invention provides a nucleotide for reducing PCR non-specific amplification, wherein the nucleotide comprises a sequence A and a sequence B, the sequence A comprises at least 6 nucleotides, and the nucleotide in the sequence A is formed by selecting three bases from A, T, C and G; the sequence B comprises at least 4 nucleotides, the nucleotides of the sequence B can be any base, and the sequence B can form a three-dimensional structure. By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and the nonspecific amplification existing in the PCR amplification process is obviously controlled.
Preferably, for the nucleotides as described above, further comprising: pretreating the nucleotide, wherein the pretreatment method comprises the following steps: connecting the sequence A with the sequence B to obtain combined nucleotide; adding the combined nucleotide into a buffer solution, heating to 65-85 ℃, preserving heat for 5-10 minutes, then cooling to 0-40 ℃, and preserving heat for 20-30 minutes. By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and nonspecific amplification in the PCR amplification process is obviously controlled, such as primer dimer reduction and the like; the primers can be promoted to rapidly enter the stable state of the polymerase chain reaction.
Preferably, for the nucleotides as described above, further comprising: the buffer solution comprises the following components: 300+ -10 mM NaCl, 5 + -1 mM MgCl 220. + -. 5 mM Tris (pH 7.6). By processing the technical parameters, the polymerase chain reaction can be completed, a target gene sequence is detected, and nonspecific amplification in the PCR amplification process is obviously controlled, such as primer dimer reduction and the like; the primers can be promoted to rapidly enter the stable state of the polymerase chain reaction.
The beneficial effects created by the invention are as follows:
the invention provides a nucleotide for reducing PCR non-specific amplification and a design method and application thereof, which can complete polymerase chain reaction, detect a target gene sequence and obviously control the non-specific amplification in the PCR amplification process, such as reducing primer dimer and the like.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to these drawings without inventive effort.
FIG. 1, a schematic diagram of the structure of a partial sequence of nucleotides;
FIG. 2 is a schematic diagram showing the structure of a partial nucleotide sequence;
FIG. 3 is a schematic diagram showing the structure of a partial nucleotide sequence;
FIG. 4, comparison of the PCR amplification of the nucleotide prepared in example 1 with the primer designed according to the conventional scheme;
FIG. 5. commercial primer v.s. qPCR amplification results for nucleotides of this example (SYBR green method).
Detailed Description
The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer.
The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, the three-dimensional structure mentioned herein includes, but is not limited to, one or more of a quadruplex DNA molecule, a G-quadruplex, a hairpin structure, if not specifically indicated.
In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments, but the invention includes but is not limited to the embodiments.
Example 1: design method of nucleotide for reducing PCR (polymerase chain reaction) nonspecific amplification
This example provides a design method for reducing non-specifically amplified nucleotides in PCR, comprising the following steps:
1. the cloning and analysis for reducing the full length of the nucleotide amplified non-specifically by PCR
(1) Design of nucleotides that reduce PCR non-specific amplification: the nucleotide for reducing the non-specific amplification of the PCR comprises a sequence A and a sequence B, wherein the sequence A comprises at least 6 nucleotides, and the nucleotides in the sequence A are formed by selecting three bases from A, T, C and G. The sequence B may form a three-dimensional structure other than a hairpin structure, the sequence B including at least 4 nucleotides, and the nucleotides of the sequence B may be any base.
In this example, the sequence A is ATGAGTATGAATTGG (SEQ ID NO: 1) and the sequence B is CCTGGGGGAGTATTGCGGAGGAAGG (SEQ ID NO: 2). The sequences of the sequence A and the sequence B were ligated together and synthesized by Competition Biotechnology engineering (Shanghai) Ltd.
The synthetic nucleotide was processed as follows: adding the synthesized nucleotide sequence into a buffer solution R, heating to 65-85 ℃, preserving heat for 5 minutes, then cooling to 0-40 ℃, preserving heat for 20-30 minutes, and obtaining the full length of the nucleotide sequence for reducing the non-specific amplification of the PCR; wherein, the buffer solution R comprises the following components: 300 mM NaCl, 5 mM MgCl2, 20 mM Tris (pH 7.6)。
The structure of the nucleotide partial sequence was determined by analysis of X single crystal diffraction data, as shown in FIG. 1.
Example 2: design method of nucleotide for reducing PCR (polymerase chain reaction) nonspecific amplification
This example provides a design method for reducing non-specifically amplified nucleotides in PCR, which differs from example 1 in that the sequence A is ACGAGCACGAACCGG (SEQ ID NO: 3) and the sequence B is GGGUUGGGAAGAAACUGUGGCACUUCGGUGCCAGCAACCC (SEQ ID NO: 4).
The synthetic nucleotide was processed as follows: adding the synthesized nucleotide sequence into a buffer solution R, heating to 65-85 ℃, preserving heat for 5 minutes, then cooling to 0-40 ℃, preserving heat for 20-30 minutes, and obtaining the full length of the nucleotide sequence for reducing the non-specific amplification of the PCR; wherein, the buffer solution R comprises the following components: 300 mM NaCl, 5 mM MgCl2, 20 mM Tris (pH 7.6).
The structure of the nucleotide partial sequence was determined by analysis of X single crystal diffraction data, as shown in FIG. 2.
Example 3: design method of nucleotide for reducing PCR (polymerase chain reaction) nonspecific amplification
This example provides a design method for reducing non-specifically amplified nucleotides in PCR, which differs from that of example 1 in that the sequence A is TCGTGC (SEQ ID NO: 5) and the sequence B is CCGG (SEQ ID NO: 6).
The synthetic nucleotide was processed as follows: adding the synthesized nucleotide sequence into a buffer solution R, heating to 65-85 ℃, preserving heat for 5 minutes, then cooling to 0-40 ℃, preserving heat for 20-30 minutes, and obtaining the full length of the nucleotide sequence for reducing the non-specific amplification of the PCR; wherein, the buffer solution R comprises the following components: 300 mM NaCl, 5 mM MgCl2, 20 mM Tris (pH 7.6).
The structure of the nucleotide partial sequence was determined by analysis of X single crystal diffraction data, as shown in FIG. 3.
Example 4: preparation method of solution for reducing PCR (polymerase chain reaction) nonspecific amplification
To the nucleotide for reducing PCR non-specific amplification prepared in example 1, 0.001. mu.M of pyridone azo compound (prepared by the method described in patent CN 105985286A) was added to prepare a solution for reducing PCR non-specific amplification.
Example 5: preparation method of solution for reducing PCR (polymerase chain reaction) nonspecific amplification
To the nucleotide for reducing PCR non-specific amplification prepared in example 1, 0.003. mu.M of a pyridone azo compound was added to prepare a solution for reducing PCR non-specific amplification.
Example 6: preparation method of solution for reducing PCR (polymerase chain reaction) nonspecific amplification
To the nucleotide for reducing PCR non-specific amplification prepared in example 1, 0.002. mu.M of pyridone azo compound was added to prepare a solution for reducing PCR non-specific amplification.
Example 7: comparative test of nucleotide and general primer designed by the invention
The PCR contrast test of the target product was performed by comparing the nucleotide for reducing non-specific amplification of PCR prepared in example 1 with the commonly used primers, using human genomic DNA as a template, and the sizes of the amplification products were 100bp, respectively, to obtain the experimental results shown in FIG. 4, which indicates that non-specific amplification did not occur when the amplification of the target product was performed using the nucleotide for reducing non-specific amplification of PCR prepared in example 1.
The amplification product is a primer sequence of a 100bp system:
S-F：CCCTGGGCTCTGTAAAGAATAGCA（SEQ ID NO：7）；
S-R：ATCAGAGCTTAAACTGGGAAGCTA（SEQ ID NO：8）。
the PCR reaction system is as follows:
the PCR thermal cycling conditions of the amplification product of the 100bp system are as follows: at 95 ℃ for 30 s;
30cycles*(95℃，15s；58℃，15s；72℃，30s)；72℃，5min。
the results of agarose gel electrophoresis are shown in FIG. 4, and the results are shown by comparing the intensities of the bands of the amplified products.
As shown in FIG. 4, Primer mix2 is the nucleotide prepared in example 1 to reduce non-specific amplification of PCR, and Primer mix1 is a Primer designed according to a conventional protocol. The experimental result shows that the non-specific amplification product is greatly reduced by the nucleotide for reducing the non-specific amplification of the PCR prepared in example 1, while the signal intensity of the specific band of the primer designed by the traditional scheme is relatively weak, and the analysis of the inventor is that the substrate is competed by the non-specific product in the reaction process. It can be seen that the nucleotide prepared in example 1 for reducing non-specific amplification in PCR amplification has sensitivity significantly better than that of the conventional method, and no impurity band appears.
In some examples of the present invention, the experiment of example 7 was repeated using the nucleotides prepared in examples 2 to 6, and again, non-specific amplification did not occur.
Example 8: comparative test of nucleotide and general primer designed by the invention
The nucleotides prepared in examples 1 to 6 for reducing non-specific amplification by PCR were compared with the commonly used primers, and PCR comparative experiments for the target products were performed, and qPCR amplification was performed under blank template conditions. The logarithm of the variation was 24 pairs (purchased from ThermoFisher Co., Ltd., product No. 4476135) of the designed primers (total concentration of primers: 5. mu.M), and DNA polymerase was amplified using the nucleotides for reducing PCR nonspecific amplification and the commercially available primers described in example 1, respectively, under the same conditions as the other reactions. And (4) carrying out agarose gel electrophoresis after the amplification is finished, and observing an electrophoresis result to show whether the amplification is successful.
The PCR reaction system is as follows: 2. mu.L (5. mu.M) of the specific primer mixture; 50ng of DNA template/quality control material; 2 XDNA polymerase working solution (DNA polymerase dosage is 0.5U) 12.5. mu.L; nuclease free water made up the volume to 25 μ L.
And (3) PCR reaction conditions:
(1) activating at 98 deg.C for 5 min; circulating for 1 time;
(2) denaturation at 98 ℃ for 15 s; annealing at 62 deg.C for 30s-10 min; extending for 2min at 72 ℃; (cycle 30 times);
(3) extending for 10min at 72 ℃; the cycle was 1 time.
Note: annealing time increases with increasing primer pair number
As shown in FIG. 5, when the primer pair number is 24 pairs, the non-specific amplification of the commercial primers is very obvious already at 30 cycles; while the non-specifically amplified nucleotides of the reduced PCR of example 1 did not show any non-specific amplification at 100 cycles.
In some examples of the present invention, the experiment of example 7 was repeated using the nucleotides prepared in examples 2 to 6, and again, non-specific amplification did not occur.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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Claims (10)
1. A design method for reducing the nucleotide which is not specifically amplified by PCR is characterized in that the nucleotide which is not specifically amplified by PCR comprises a sequence A and a sequence B, wherein the sequence A comprises at least 6 nucleotides, and the nucleotide in the sequence A selects three bases from A, T, C and G as the structure; the sequence B comprises at least 4 nucleotides, the nucleotides of the sequence B can be any base, and the sequence B can form a three-dimensional structure.
2. The design method according to claim 1, wherein the nucleotides for reducing non-specific amplification of PCR are subjected to a pretreatment comprising: connecting the sequence A with the sequence B to obtain combined nucleotide; adding the combined nucleotide into a buffer solution, heating to 65-85 ℃, preserving heat for 5-10 minutes, then cooling to 0-40 ℃, and preserving heat for 20-30 minutes.
3. The design method according to claim 2, wherein the buffer has a composition of: 300+ -10 mM NaCl, 5 + -1 mM MgCl2, 20±5 mM Tris (pH 7.6)。
4. The method of claim 2, wherein the buffer comprises 0.001 to 0.003 μ M of a pyridone azo compound.
5. A nucleotide prepared by the design method according to any one of claims 1 to 4.
6. Use of the nucleotide of claim 5 in PCR amplification.
7. The application of claim 6, wherein the application comprises: in the PCR amplification, DNA polymerase and 0.001-0.003 mu M pyridone azo compound are used.
8. A nucleotide for reducing non-specific amplification of PCR, wherein the nucleotide comprises a sequence A and a sequence B, the sequence A comprises at least 6 nucleotides, and the nucleotide in the sequence A is formed by selecting three bases from A, T, C and G; the sequence B comprises at least 4 nucleotides, the nucleotides of the sequence B can be any base, and the sequence B can form a three-dimensional structure.
9. The nucleotide of claim 8, wherein the nucleotide is pre-treated by a pre-treatment method comprising: connecting the sequence A with the sequence B to obtain combined nucleotide; adding the combined nucleotide into a buffer solution, heating to 65-85 ℃, preserving heat for 5-10 minutes, then cooling to 0-40 ℃, and preserving heat for 20-30 minutes.
10. The nucleotide according to claim 8 or 9, wherein the buffer has the composition: 300+ -10 mM NaCl, 5 + -1 mM MgCl2, 20±5 mM Tris (pH 7.6)。
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104011224A (en) * | 2011-12-22 | 2014-08-27 | 霍夫曼-拉罗奇有限公司 | Methods and reagents for reducing non-specific amplification |
| CN104093850A (en) * | 2012-02-15 | 2014-10-08 | 通用电气公司 | Methods and kits for reducing non-specific nucleic acid amplification |
| WO2016137031A1 (en) * | 2015-02-25 | 2016-09-01 | (주)다이오진 | Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same |
| CN107849603A (en) * | 2015-04-24 | 2018-03-27 | 阿提拉生物系统公司 | Expanded using the primer of limited nucleotides composition |
| CN108107028A (en) * | 2018-02-02 | 2018-06-01 | 济南大学 | A kind of detection atriphos（ATP）Biosensor |
| CN111183229A (en) * | 2017-08-11 | 2020-05-19 | 阿提拉生物系统股份有限公司 | Digital amplification using primers with limited nucleotide composition |
| CN112538482A (en) * | 2019-09-23 | 2021-03-23 | 华东理工大学 | RNA detection and quantification method |
| CN112986551A (en) * | 2019-12-11 | 2021-06-18 | 清华大学 | Method and kit for detecting concentration of target molecules in mixed system |
-
2021
- 2021-01-05 CN CN202110006795.7A patent/CN112899346A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104011224A (en) * | 2011-12-22 | 2014-08-27 | 霍夫曼-拉罗奇有限公司 | Methods and reagents for reducing non-specific amplification |
| CN104093850A (en) * | 2012-02-15 | 2014-10-08 | 通用电气公司 | Methods and kits for reducing non-specific nucleic acid amplification |
| WO2016137031A1 (en) * | 2015-02-25 | 2016-09-01 | (주)다이오진 | Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same |
| CN107075576A (en) * | 2015-02-25 | 2017-08-18 | 精确诊断有限公司 | Dumbbell structure oligonucleotides, the nucleic acid amplification primers comprising it and the nucleic acid amplification method using the primer |
| CN107849603A (en) * | 2015-04-24 | 2018-03-27 | 阿提拉生物系统公司 | Expanded using the primer of limited nucleotides composition |
| CN111183229A (en) * | 2017-08-11 | 2020-05-19 | 阿提拉生物系统股份有限公司 | Digital amplification using primers with limited nucleotide composition |
| CN108107028A (en) * | 2018-02-02 | 2018-06-01 | 济南大学 | A kind of detection atriphos（ATP）Biosensor |
| CN112538482A (en) * | 2019-09-23 | 2021-03-23 | 华东理工大学 | RNA detection and quantification method |
| CN112986551A (en) * | 2019-12-11 | 2021-06-18 | 清华大学 | Method and kit for detecting concentration of target molecules in mixed system |
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