CN116064750A - Reaction system for multiplex PCR amplification and amplification kit thereof - Google Patents
Reaction system for multiplex PCR amplification and amplification kit thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a reaction system for multiplex PCR amplification and an amplification kit thereof, in particular to the multiplex PCR amplification reaction system disclosed by the invention, which can improve the equalization, compatibility, specificity and sensitivity of multiplex PCR amplification; fragments of greater difference in length in the same multiplex reaction can be amplified efficiently. Has application significance for various practical applications of multiplex amplification, in particular to an amplicon library-building reaction system in second generation sequencing.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a reaction system for multiplex PCR amplification and an amplification kit thereof.
Background
The Polymerase Chain Reaction (PCR) is a molecular biological technique that uses two oligonucleotides as primers and that uses a DNA polymerase to catalyze the amplification of a DNA fragment located between the two primers. Since the establishment of the technology in 1983, the technology has become a main body and a key method in the current life science research and related fields because of the characteristics of high sensitivity, high efficiency and high specificity. A series of related techniques such as nested PCR, real-time quantitative PCR, immuno-PCR, multiplex PCR, etc. have been developed. Wherein, the multiplex PCR rapidly permeates into various fields of life science, such as amplicon library establishment, microsatellite analysis, genotyping, SNP detection and the like, due to the advantages of high specificity, high efficiency and low cost.
Multiplex PCR (multiplex PCR) is developed based on conventional PCR, and multiplex PCR is improved based on conventional PCR, and a PCR technology for amplifying a plurality of target fragments by adding a plurality of pairs of specific primers to a PCR reaction system and aiming at a plurality of DNA templates or different regions of the same template. The principle, reagent and operation process of multiplex PCR are the same as that of conventional PCR, except that 2 pairs or more than 2 pairs of primers are added into the multiplex PCR reaction system, and a plurality of target sites are amplified simultaneously, namely different target sites of one or more genes can be amplified simultaneously in one-tube PCR reaction. Thus, multiple target sequences can be amplified by a single PCR using a primer set capable of amplifying each target sequence. Compared with single PCR, multiple PCR has the advantages of high detection efficiency and cost saving. In general, multiplex PCR can obtain the same sensitivity as that of single PCR in the range of 4-5 pairs of primers, and is difficult to optimize for more than 5 pairs of PCR, and the specificity is greatly reduced, so that non-specific products are generated, and multiplex PCR of more than 20 pairs of primers is difficult to avoid non-specific reaction even if a large number of primer sequences are screened, and the amplification cycle number is limited to a certain range, but such multiplex PCR has lower sensitivity. Therefore, those skilled in the art are working to improve the sensitivity, stability, etc. of multiplex PCR reactions to meet the needs of different detection fields.
Disclosure of Invention
The invention aims to provide an amplification reaction system for multiplex PCR and an amplification kit thereof, which are applied to multiple fields of multiplex PCR amplification and can realize specific and efficient amplification of multiple target sites.
In a first aspect of the present invention, there is provided a PCR reagent comprising:
Tris-HCl, magnesium chloride, potassium chloride, ammonium sulfate, trehalose, beta-cyclodextrin, DMSO, BSA, tween and water.
In another preferred embodiment, the content of each component in the PCR reagent comprises, in parts by unit:
the balance being water.
In another preferred embodiment, the PCR reagent comprises:
the balance being water.
In another preferred embodiment, the pH of the PCR reagent is 8.0-9.0; preferably 8.3-8.8; more preferably 8.6.
In another preferred embodiment, the PCR reagents comprise:
the balance being water.
In another preferred embodiment, the PCR reagents comprise:
the balance being water.
In another preferred embodiment, the PCR reagent further comprises dNTPs.
In another preferred embodiment, the dNTPs have the following composition ratio: dATP: dTTP: dCTP: dgtp=1: 1:1:1.
in a second aspect of the present invention, there is provided a multiplex PCR amplification reaction system comprising a PCR reagent according to the first aspect of the present invention.
In another preferred embodiment, the multiplex PCR amplification is a PCR amplification of 3 or more; preferably not less than 5 weight by PCR; more preferably not less than 8 weight by PCR; more preferably PCR amplification of ≡10 weight; more preferably 12% by weight PCR amplification; more preferably a PCR amplification of 15 or more; more preferably not less than 20 by weight.
In a third aspect of the invention, there is provided the use of a PCR reagent according to the first aspect of the invention in the preparation of a multiplex PCR amplification reaction system or a multiplex PCR amplification kit.
In a fourth aspect of the invention there is provided a multiplex PCR amplification kit comprising a PCR reagent according to the first aspect of the invention.
In another preferred embodiment, the multiplex PCR amplification kit further comprises one or more components selected from the group consisting of: DNA polymerase, primer, DNA template, and ddH 2 O。
In a fifth aspect of the present invention, there is provided a multiplex PCR amplification method comprising the steps of:
(1) Preparation of multiplex PCR amplification System
The multiplex PCR amplification system comprises: the PCR reagent, the DNA polymerase and the multiplex primer of the first aspect of the invention;
(2) Multiplex PCR amplification reactions were performed.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a diagram of Qseq 100 full-automatic nucleic acid protein analyzer assay of experimental group of example 1.
FIG. 2 is a diagram of Qseq 100 full-automatic nucleic acid protein analyzer assay for control group of example 1.
FIG. 3 is a chart of multiple amplified agarose gel electrophoresis of the experimental group of example 2 at different template concentrations.
FIG. 4 is a chart showing the agarose gel electrophoresis of the multiplex amplification of the control group of example 2 at different template concentrations.
FIG. 5 is a chart of agarose gel electrophoresis of multiple amplifications of fragments of interest of different lengths for the experimental set of example 3.
FIG. 6 is a chart of agarose gel electrophoresis of multiple amplifications of fragments of interest of different lengths for the control group of example 3.
FIG. 7 is a graph showing the results of the blood card direct expansion of the experimental group of example 4.
FIG. 8 is a graph showing the results of the blood card direct expansion of the control group of example 4.
Major reagents and instrumentation:
instrument: 200ul EP tube, 1.5ml EP tube, vortex shaker, transient centrifuge, PCR instrument reagents: tris, magnesium chloride, potassium chloride are produced by VWR corporation;
ammonium sulfate, trehalose, beta-cyclodextrin, BSA (bovine serum albumin), tween 20, manufactured by aladin company.
Detailed Description
Through intensive researches, the invention provides a multiplex PCR amplification reaction system, which can improve the balance, compatibility, specificity and sensitivity of multiplex PCR amplification and can perform efficient amplification on more than 5 weight PCR; fragments with larger length difference in the same multiplex reaction can be effectively amplified, so that the method not only can be used for amplifying short fragments and multi-site target products, but also can be used for amplifying long fragment target products in multiplex PCR reaction, and the length range of amplified products is wide. Has application significance for various practical applications of multiplex amplification, in particular to an amplicon library-building reaction system in second generation sequencing.
The invention is realized by adopting the following technical scheme.
In a preferred embodiment of the present invention, the present invention provides a multiplex PCR amplification reaction system consisting of, in parts by unit:
pH8.0-9.0, the balance being water, beta-cyclodextrin being mass volume percent, DMSO, tween 20 being volume percent.
Preferably, the reaction system consists of the following components in parts by unit:
pH8.3-8.8, the balance being water, beta-cyclodextrin being mass volume percent, DMSO, tween 20 being volume percent.
Preferably, the reaction system consists of the following components in parts by unit:
pH8.6, the balance being water, beta-cyclodextrin as mass volume percentage, DMSO, tween 20 as volume percentage.
In the present invention, "in unit parts" means the content of each active ingredient (active ingredient is Tris-HCl, magnesium chloride, potassium chloride, ammonium sulfate, trehalose, beta-cyclodextrin, DMSO, BSA, and Tween 20) in one PCR amplification reaction system at the time of use. In other embodiments, the PCR reagents of the invention may be formulated as desired in a concentrated form, e.g., the concentration of each active ingredient is increased by the same factor, simply by dilution to the desired concentration at the time of use.
In another preferred embodiment of the present invention, the present invention also provides a multiplex PCR amplification kit comprising any of the reaction systems described above;
in a preferred embodiment of the present invention, in the multiplex PCR amplification kit provided by the present invention, dNTPs are added to the PCR reaction solution of the reaction system, and the composition ratio of dNTPs is as follows: dATP: dTTP: dCTP: dgtp=1: 1:1:1, preferably at a concentration of 0.3mM.
Preferably, the kit further comprises: DNA polymerase, detection primer, DNA template, ddH 2 O。
The invention has the main advantages that:
(1) The multiplex PCR amplification reaction system can improve the equalization, compatibility, specificity and sensitivity of multiplex PCR amplification.
(2) The multiplex PCR amplification reaction system of the invention can be used for carrying out high-efficiency amplification on more than 5 weight PCR.
(3) The multiplex PCR amplification reaction system can be used for effectively amplifying fragments with larger length difference in the same multiplex reaction, can be used for amplifying short fragments and multi-site target products, can also be used for amplifying long fragment target products in the multiplex PCR reaction, and has wide length range of amplified products.
(4) The multiplex PCR amplification reaction system has application significance for various practical applications of multiplex amplification, in particular to an amplicon library-building reaction system in second-generation sequencing.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Example 1
1. Human fresh blood DNA was extracted as an amplification template by conventional methods.
The experimental group reaction system (1×) was as follows: tris-HCl 50mM, magnesium chloride 2.5mM, potassium chloride 35mM, ammonium sulfate 20mM, trehalose 0.15M, beta-cyclodextrin 3% (w/v), DMSO 5% (v/v), BSA 0.3mg/ml, tween 20.15% (v/v), dNTPs 0.3mM, pH8.6, and formulated into 2X PCR Master Mixture-1 for use.
The control reaction system (1×) was as follows: tris-HCl 50mM, magnesium chloride 2.5mM, potassium chloride 35mM, ammonium sulfate 20mM, trehalose 0.15M,Tween 20 0.15% (v/v), dNTPs 0.3mM, pH8.6, were prepared to 2X PCR Master Mixture-1 for use.
The concentration of the human genome template is 2.5ng/ul, and the input amount is 10ng.
2. The primer used in the system is 14 pairs, which are 14 sites for amplifying the driving genes related to human lung cancer, and the primer sequences are shown in table 1. Wherein the molar ratio of each primer pair is as follows: MET-1: MET-2: PIK3CA-1: PIK3CA-2: RET-1: RET-2: ERBB2: BRAF: ROSI-1: ROSI-2: ROSI-3: ROSI-4: ROSI-5: ROSI-6=0.1: 0.2:0.25:0.2:0.2:0.1:0.1:0.1:0.08:0.08:0.1:0.1:0.1:0.1.
TABLE 1 multiplex PCR primer sequence 1
| Primer name | Nucleotide sequence | SEQ ID NO. | |
| MET- | GGTTTGATAAATAATTATTTC | 1 | |
| MET- | ATAAAATGCCACTTACTG | 2 | |
| MET- | AAGGTTGCTGATTTTGGTCTTG | 3 | |
| MET- | TTGGTGGTAAACTTTTGAG | 4 | |
| PIK3CA- | GCCATTGACCTGTTTACACG | 5 | |
| PIK3CA- | AAACAGAGAAAACCATTAC | 6 | |
| PIK3CA- | GAACTACAATCTTTTGATGCA | 7 | |
| PIK3CA- | GTTTAATTGTGTGGAAGATCC | 8 | |
| RET- | ACCTGGTATGGTCATGGAA | 9 | |
| RET- | ATGTGGGTGGTTGACCTGCT | 10 | |
| RET- | AGCTCGTTCATCGGGACTTG | 11 | |
| RET- | CCATGGTGCACCTGGGAT | 12 | |
| ERBB2- | GGAGGCTGTGTGGTGTTTG | 13 | |
| ERBB2- | TCACCAGCTGCACCGTGGAT | 14 | |
| BRAF-F | GATCCAGACAACTGTTCA | 15 | |
| BRAF-R | TCTTCATAATGCTTGCTCTC | 16 | |
| ROSI-1F | AAATCAACCCATTTCCTCACC | 17 | |
| ROS1-1R | CCTCAGAGCTAGAGCGATTG | 18 | |
| ROS1-2F | TCGCTTGTATTGGACAGAAGTT | 19 | |
| ROS1- | CGAGCATAGCAGGTACTGTGA | 20 | |
| ROS1-3F | AACAGCAGGAAAAACCCTTG | 21 | |
| ROS1-3R | CCTGGAAAAGGCTGCATAAC | 22 | |
| ROS1-4F | AAGCGTTTCTGTCTCTAGCTTCA | 23 | |
| ROS1-4R | GACCACTAGTTCCAATCTTACCAGA | 24 | |
| ROS1-5F | TTTGCAGAAAGAGCACTTCAAA | 25 | |
| ROS1-5R | CAGACATGGTAACATACCTCCAA | 26 | |
| ROS1-6F | ACTTCTGCCACAGGGATCTG | 27 | |
| ROS1-6R | TGTGTCCCGTTAAACTTACCA | 28 |
3. The multiplex amplification reaction was 20ul in total and included:
0.4ul of DNA polymerase, 10ul of reaction system (2X PCR Master Mixture-1); primer 4ul; DNA 4ul (2.5 g/ul); ddH2O 1.6ul.
4. The PCR reaction procedure was 95℃for 2min; (95 ℃ C. 10s,60 ℃ C. 1min 30 s), 22 cycles; 72 ℃ for 5min; hold at 4 ℃.
The multiplex amplification products are purified by using a magnetic bead method DNA purification recovery kit of Jiangsu kang which is century biotechnology Co., ltd according to the method of the specification, and the concentration of the products is measured by using a Qubit3 fluorescent agent, and the products are detected by using a Qseq 100 full-automatic nucleic acid protein analyzer.
As shown in the figure 1, the experimental group detection results show that the product obtained by using the multiplex PCR amplification reaction system has high specificity and good balance.
As shown in FIG. 2, the control group showed poor uniformity and poor specificity of the products obtained by using the multiplex PCR amplification reaction system.
Example 2
1. And detecting by taking lambda DNA as an amplification template.
The experimental group reaction system (1×) was as follows: tris-HCl 50mM, magnesium chloride 3mM, potassium chloride 50mM, ammonium sulfate 20mM, trehalose 0.1M, beta-cyclodextrin 3% (w/v), DMSO 5% (v/v), BSA 0.3mg/ml, tween 20.15%, dNTPs 0.3mM, pH8.6, and 2X PCR Master Mixture-2.
The control reaction system (1×) was as follows: tris-HCl 50mM, magnesium chloride 3mM, potassium chloride 50mM, ammonium sulfate 20mM, beta-cyclodextrin 3% (w/v), DMSO 5% (v/v), dNTPs 0.3mM, pH8.6, and formulated into 2X PCR Master Mixture-2 for use.
The amounts of lambda DNA template added were 1ng, 100pg, 10pg, and 1pg, respectively, and lambda DNA templates of different concentrations were prepared according to the amounts added.
2. The number of primers used in the system is 8, and the sequences of the primers are shown in Table 2. Wherein the molar ratio of each primer pair is as follows: 0.2:0.2:0.2:0.15:0.1:0.1:0.1:0.18.
TABLE 2 multiplex PCR primer sequence 2
| Primer name | Nucleotide sequence | SEQ ID NO. | |
| IP-2F | AAGCAGCTGGCTGACATTTT | 29 | |
| IP- | AGCATCCCTTTCGGCATAC | 30 | |
| IP-4F | GTGAAAAGTCGGTGGATGTG | 31 | |
| IP-4R | CATAAAATGCGGGGATTCAC | 32 | |
| IP-5F | ACATCCGTGAGGTGAATGTG | 33 | |
| IP-5R | GTGAAACGCTTCATGGTGAG | 34 | |
| IP-6F | AGCAGCTGGCTGACATTTTC | 35 | |
| IP-6R | CGGAGTCTCTGGCATTCTTC | 36 | |
| IP-8F | GTGGCAAGGGTAATGAGGTG | 37 | |
| IP-8R | ATCAACATGTCGGTTTTCCAG | 38 | |
| IP-9F | GTGAATCCCCGCATTTTATG | 39 | |
| IP-9R | AGCGTGGTGTTTACGAAGGT | 40 | |
| IP-10F | TAACACGCTCACCATGAAGC | 41 | |
| IP-10R | CGGGCATCAGTAAAGTCCAG | 42 | |
| IP-12F | TTCCTGGGTGACAAGCGTAT | 43 | |
| IP-12R | AGCGTGGTGTTTACGAAGGT | 44 |
3. The multiplex amplification reaction was 20ul in total and included: DNA polymerase 0.4ul10ul of a reaction system (2X PCR Master Mixture-2); primer 4ul; DNA 2ul; ddH 2 O 3.6ul。
The PCR reaction procedure was 95℃for 2min; (95 ℃ C. 10s,60 ℃ C. 4 min), 22 cycles; 72 ℃ for 5min; hold at 4 ℃.
Multiplex amplification products were detected by 3% agarose gel electrophoresis.
The test results of the test group are shown in FIG. 3, and the results show that the reaction system provided by the invention can reach the lower limit of detection of 1pg (lambda DNA system), which can prove that the reaction system provided by the invention has higher amplification sensitivity.
The detection results of the control group are shown in FIG. 4, and the results show that the amplification sensitivity of the control group reaction system is 10pg.
Example 3
1. The detection is carried out by taking the human genome as a template.
The experimental group reaction system (1×) was as follows: tris-HCl 50mM, magnesium chloride 2.5mM, potassium chloride 30mM, ammonium sulfate 20mM, trehalose 0.15M, beta-cyclodextrin 3% (w/v), DMSO 5% (v/v), BSA 0.3mg/ml, tween 20.15% (v/v), dNTPs 0.3mM, pH8.6, and formulated into 2X PCR Master Mixture-3 for use.
The control reaction system (1×) was as follows: tris-HCl 50mM, magnesium chloride 2.5mM, potassium chloride 30mM, ammonium sulfate 20mM, beta-cyclodextrin 3%, BSA 0.3mg/ml, tween 20.15%, dNTPs 0.3mM, pH8.6, and formulated into 2X PCR Master Mixture-3 for use.
The concentration of the human genome template is 10ng/ul, and the input amount of the DNA template is 20ng.
2. The primer used in the system is 3 pairs, and the primer sequences are 287bp, 1197bp and 2286bp respectively as shown in the table 3. Wherein the molar ratio of each primer pair is as follows: ROS1-6: AZU1: hbb=0.2: 0.1:0.2.
TABLE 3 multiplex PCR primer sequence 3
| Primer name | Nucleotide sequence | SEQ ID NO. |
| ROS1-6F | ACTTCTGCCACAGGGATCTG | 45 |
| ROS1-6R | TGTGTCCCGTTAAACTTACCA | 46 |
| AZU1-F | CTCTCATTCATGGGGCCCTC | 47 |
| AZU1-R | CATTGGGTGAGGTGGCCA | 48 |
| HBB-F | GATGAAGTTGGTGGTGAGGC | 49 |
| HBB-R | CATGATAAGGGAGCCAGCAG | 50 |
3. The multiplex amplification reaction was 20ul in total and included: 0.4ul of DNA polymerase, 10ul of reaction system (2X PCR Master Mixture-3); primer 5ul; DNA 2ul; ddH 2 O 2.6ul。
The PCR reaction procedure was 95℃for 2min; (95 ℃ C. 10s,60 ℃ C. 4 min), 22 cycles; 72 ℃ for 10min; hold at 4 ℃.
Multiplex amplification products were detected by 2% agarose gel electrophoresis. The test results of the experimental group and the control group are shown in fig. 5 and 6, respectively, in which:
m: DNA molecular weight markers
1. 14: triple PCR
2-4, 11-13: dual PCR
5-7, 8-10: single PCR
The results of FIG. 5 show that the reaction system of the invention can amplify fragments with larger length difference, which shows that the reaction system of the invention can be used for amplifying short fragments and multi-site target products, and can also be used for amplifying long-fragment target products in multiple PCR reactions, and the length range of amplified products is wide.
The results in FIG. 6 show that the control reaction system cannot amplify specific target fragments and is at risk of missed detection.
Example 4
1. And detecting by taking the blood card as a template.
The experimental group reaction system is as follows: tris-HCl 50mM, magnesium chloride 3mM, potassium chloride 50mM, ammonium sulfate 25mM, trehalose 0.15M, beta-cyclodextrin 4% (w/v), DMSO 5% (v/v), BSA 0.6mg/ml, tween 20.15% (v/v), dNTPs 0.3mM, pH 8.8, are formulated into 2X PCR Master Mixture-4 for use.
The control reaction system was as follows: tris-HCl 50mM, magnesium chloride 3mM, potassium chloride 50mM, ammonium sulfate 25mM, beta-cyclodextrin 4% (w/v), DMSO 5% (v/v), tween 20.15% (v/v), dNTPs 0.3mM, pH 8.8, and formulated into 2X PCR Master Mixture-4 for use.
2. The multiplex amplification reaction was 20ul in total and included: 0.4ul of DNA polymerase, 10ul of reaction system (2X PCR Master Mixture-4); primer 5ul; the diameter of the blood card template sample is 1mm; ddH 2 O 4.6ul。
TABLE 4 multiplex PCR primer sequence 4
The PCR reaction procedure was 95℃for 2min; (95 ℃ C. 10s,60 ℃ C. 1min 30 s), 28 cycles; 15min at 60 ℃; hold at 4 ℃.
The multiplex amplification products were detected by capillary electrophoresis using a 3130xl fragment analyzer.
The results of the experimental group are shown in FIG. 7, and the results show that the reaction system of the invention has excellent tolerance in blood card multiplex PCR amplification.
The results of the control group are shown in FIG. 8, and the results show that the reaction system of the control group has poor tolerance in blood card multiplex PCR amplification.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. A PCR reagent, wherein the PCR reagent comprises:
Tris-HCl, magnesium chloride, potassium chloride, ammonium sulfate, trehalose, beta-cyclodextrin, DMSO, BSA, tween and water.
2. The PCR reagent of claim 1, wherein the content of each component of the PCR reagent comprises, in parts by unit:
the balance being water.
3. The PCR reagent of claim 1, wherein the pH of the PCR reagent is 8.0-9.0; preferably 8.3-8.8; more preferably 8.6.
4. The PCR reagent of claim 2, wherein the PCR reagent comprises:
the balance being water.
5. The PCR reagent of claim 1, wherein the PCR reagent further comprises dNTPs.
6. A multiplex PCR amplification reaction system comprising the PCR reagent of claim 1.
7. The use of the PCR reagent of claim 1 in the preparation of a multiplex PCR amplification reaction system or a multiplex PCR amplification kit.
8. A multiplex PCR amplification kit comprising the PCR reagent of claim 1.
9. The multiplex PCR amplification kit of claim 8, further comprising one or more components selected from the group consisting of: DNA polymerase, primer, DNA template, and ddH 2 O。
10. A multiplex PCR amplification method, comprising the steps of:
(1) Preparation of multiplex PCR amplification System
The multiplex PCR amplification system comprises: the PCR reagent, DNA polymerase, and multiplex PCR primer of claim 1;
(2) Multiplex PCR amplification reactions were performed.
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