CN113862340A - Economical and time-saving micro PCR method - Google Patents
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Abstract
The invention discloses a time-saving and time-saving micro PCR method, and belongs to the field of molecular biology. The method comprises the following sample adding steps: 1) adding the PCR premix solution to the bottom of a PCR tube; 2) diluting the DNA template with water; 3) and (3) adding all the diluted DNA templates into a PCR tube, and centrifugally mixing to obtain a PCR reaction system. Preferably, in step 3), the DNA template is added to the middle wall of the PCR tube. The method can improve the precision of PCR detection, reduce detection errors, save reagents, reduce the times of replacing pipette tips and save time and labor under a smaller-volume PCR system.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a time-saving and time-saving micro PCR method.
Background
Real-time fluorescent quantitative polymerase chain reaction, called fluorescent quantitative PCR. The detection means is based on PCR, adds a fluorescent group, and utilizes the change of a fluorescent signal to monitor the process in real time, thereby achieving the purpose of relatively quantifying the target gene. In the fluorescent quantitative PCR process, a PCR reaction system (usually more than 20. mu.l) needs to be constructed, which comprises prepared premixed liquid (enzyme mixed liquid, primers, sterilized water and the like) and a DNA template. The sample is added to the PCR reaction system by adding the prepared pre-mixed solution to the PCR tube, and adding 2. mu.l of DNA template to the pre-mixed solution.
Because the volume of the DNA template added into each PCR tube is small, large sample loading errors are easy to occur particularly when a PCR reaction system with the volume less than 20 mul, such as a PCR reaction system with the volume less than 10 mul, needs to be constructed. And because the premixed solution needs to be touched when the DNA template is added every time, a pipette tip (also called a 'gun tip') needs to be replaced when the template is loaded every time, the batch operation is time-consuming and labor-consuming, and the disposable tip is directly discarded, thereby causing great waste.
Therefore, it is very important to provide a PCR method with low cost, time and labor, and small error.
Disclosure of Invention
The invention aims to solve the problems that: provides a PCR method with small error and time and labor saving.
The technical scheme of the invention is as follows:
a method for loading a PCR reaction system, the method comprising the steps of:
1) adding the PCR premix solution to the bottom of a PCR tube;
2) diluting the DNA template by 3-6 times with water;
3) the DNA template was added to the PCR tube and mixed by centrifugation.
The sample adding method of the PCR reaction system is as described above, and in step 3), the DNA template is added to the middle tube wall of the PCR tube.
The sample adding method of the PCR reaction system is as described above, and in the step 2), the dilution ratio is 4.2 times.
According to the sample adding method of the PCR reaction system, the total volume of the PCR reaction system is 10-25 mu l.
As for the loading method of the PCR reaction system, the total volume of the PCR reaction system is 10. mu.l.
And a PCR method, wherein the PCR reaction system is prepared by using the sample adding method of the PCR reaction system.
The PCR method is the real-time fluorescent quantitative PCR.
The invention has the following beneficial effects:
1. by diluting the DNA template before sample adding, the invention can improve the precision of PCR detection and save the reagent in a smaller PCR reaction system (10 mu l).
2. According to the invention, the DNA template is added to the middle pipe wall of the PCR pipe, so that the frequency of replacing the gun tip can be reduced, the time and the labor are saved, and the gun tip is saved.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
Reagents and instruments used in the invention:
TaKaRa
Premix Ex TaqTMⅡ(Tli RNaseH Plus)(RR820Q，TaKaRa)。
DNA template: cDNA reverse-transcribed from extracted total RNA of human immortalized keratinocytes (HaCat, China Center for Type Culture Collection, 0106).
Primer: HMGB1 Forward (5 'to 3', SEQ ID NO. 1): GAGAGGTGGAAGACCAT, HMGB1 Reverse (5 'to 3', SEQ ID NO. 2): CAGAGCAGAAGAGGAAGA are provided. The primer was synthesized by Oncology Biotechnology, Inc.
8-Tube PCR Strips(Bio-Rad，TLS0851)；
Refrigerated centrifuge (Thermo, fresh co 17);
normal temperature centrifuge (Thermo, Legend Micro 17);
an eight tube centrifuge (labsic, SCILOX S1010E);
pipettor 2.5. mu.L (Eppendorf Co., 0.2-2.5. mu.L);
pipette 10. mu.L (Eppendorf Co., 1-10. mu.L);
pipette 100. mu.L (Eppendorf Co., 10-100. mu.L);
pipette 1000. mu.L (Eppendorf Co., 100-;
the PCR instrument (Roche,
)。
example 1 sample application method of PCR reaction System of the present invention
Configuring a Real time PCR reaction system:
a Real time PCR reaction system (10. mu.l) was prepared as follows:
premix Ex Taq II (2X), 5. mu.l; PCR forward primer (10. mu.M), 0.4. mu.l; PCR reverse primer (10. mu.M), 0.4. mu.l; template DNA (diluted 4.2-fold with sterile water), 4.2. mu.l.
The amount of the DNA template was calculated according to the number of wells made, and the DNA template was diluted 4.2 times with sterilized water according to the amount. And according to the number of the holes made, calculating
Premix Ex Taq II (2X) and primer content were mixed in appropriate amounts to prepare a Premix.
Premix was added to the bottom of the corresponding PCR tube, 5.8. mu.l/tube. The diluted DNA template was then added to the middle wall of the corresponding PCR tube, 4.2. mu.l/tube. When the same DNA template is added, the gun tip does not need to be replaced.
After sample addition, the PCR premix solution and the template are fully mixed by using an eight-tube centrifuge for instantaneous centrifugation.
Example 2 sample application method of PCR reaction System of the present invention
Configuring a Real time PCR reaction system:
a Real time PCR reaction system (20. mu.l) was prepared as follows:
premix Ex Taq II (2X), 15. mu.l; PCR forward primer (10. mu.M), 1. mu.l; PCR reverse primer (10. mu.M), 1. mu.l; template DNA (3-fold diluted in sterile water) in 3. mu.l.
The amount of the DNA template was calculated according to the number of wells made, and the DNA template was diluted 3-fold with sterilized water according to the amount. And according to the number of the holes made, calculating
Premix Ex Taq II (2X) and primer content were mixed in appropriate amounts to prepare a Premix.
The premix was added to the bottom of the corresponding PCR tube at 17. mu.l/tube. The diluted DNA template was then added to the middle wall of the corresponding PCR tube at 3. mu.l/tube. When the same DNA template is added, the gun tip does not need to be replaced.
After sample addition, the PCR premix solution and the template are fully mixed by using an eight-tube centrifuge for instantaneous centrifugation.
Example 3 sample application method of PCR reaction System of the present invention
Configuring a Real time PCR reaction system:
a Real time PCR reaction system (25. mu.l) was prepared as follows:
premix Ex Taq II (2X), 15. mu.l; PCR forward primer (10. mu.M), 2. mu.l; PCR reverse primer (10. mu.M), 2. mu.l; template DNA (diluted 6-fold in sterile water) was added in 6. mu.l.
The amount of the DNA template was calculated according to the number of wells made, and the DNA template was diluted 6-fold with sterilized water according to the amount. And according to the number of the holes made, calculating
Premix Ex Taq II (2X) and primer content were mixed in appropriate amounts to prepare a Premix.
Premix was added to the bottom of the corresponding PCR tube, 19. mu.l/tube. The diluted DNA template was then added to the middle wall of the corresponding PCR tube, 6. mu.l/tube. When the same DNA template is added, the gun tip does not need to be replaced.
After sample addition, the PCR premix solution and the template are fully mixed by using an eight-tube centrifuge for instantaneous centrifugation.
Experimental example 1 application and Performance test of sample application method of PCR reaction System of the present invention
1. Repeated precision measurements at different sample application locations.
1.1 Real time PCR reaction system configuration.
A Real time PCR reaction system (10. mu.l) was prepared as follows:
premix Ex Taq II (2X), 5. mu.l; PCR forward primer (10. mu.M), 0.4. mu.l; PCR reverse primer (10. mu.M), 0.4. mu.l; template DNA (diluted 4.2-fold with sterile water), 4.2. mu.l.
The amount of the DNA template was calculated according to the number of wells made, and the DNA template was diluted 4.2 times with sterilized water according to the amount. And according to the number of the holes made, calculating
Premix Ex Taq II (2X) and primer content were mixed in appropriate amounts to prepare a Premix.
(1) Sample application position according to the present invention (example 1): premix was added to the bottom of the corresponding PCR tube, 5.8. mu.l/tube. The diluted DNA template was then added to the middle wall of the corresponding PCR tube, 4.2. mu.l/tube. When the same DNA template is added, the gun tip does not need to be replaced.
(2) Traditional loading position (as control): premix was added to the bottom of the corresponding PCR tube, 5.8. mu.l/tube. The diluted DNA template was then added to the bottom of the corresponding PCR tube, 4.2. mu.l/tube. For each PCR reaction tube, the tip of the gun was changed when the same DNA template was added.
After sample addition, the PCR premix solution and the template are fully mixed by using an eight-tube centrifuge for instantaneous centrifugation.
1.2 fluorescent quantitative PCR detection.
Performing fluorescent quantitative PCR amplification. Standard procedure for PCR amplification: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 20s, 40 cycles. Dissolution curve: 95 ℃ for 10s, 65 ℃ for 60s and 97 ℃ for 1 s.
1.3 repeated precision measurements at different sample application positions.
The different loading methods set up 6 replicates. From the measured Ct values, the Coefficient of Variation (CV) of the Ct values was calculated and compared.
1.4 intermediate precision measurement of sample application positions of different templates.
The experiments were performed 3 times at different times, and 3 replicates were performed at each time. From the measured Ct values, the Coefficient of Variation (CV) of the Ct values was calculated and compared.
2. Precision determination of sample volumes for different templates.
2.1 Real time PCR reaction system configuration.
The Real time PCR reaction system was configured as follows. Experimental group Real time PCR reaction system (10 μ l):
premix Ex Taq II (2X), 5. mu.l; PCR forward primer (10. mu.M), 0.4. mu.l; PCR reverse primer (10. mu.M), 0.4. mu.l; template DNA (diluted 4.2-fold with sterile water), 4.2. mu.l. Control Real time PCR reaction (10. mu.l):
premix Ex Taq II (2X), 5. mu.l; PCR forward primer (10. mu.M), 0.4. mu.l; PCR reverse primer (10. mu.M), 0.4. mu.l; template DNA, 1. mu.l; sterilized distilled water, 3.2. mu.l.
Experimental group Real time PCR reaction system (10 μ l): the amount of the DNA template was calculated according to the number of wells made, and the DNA template was diluted 4.2 times with sterilized water according to the amount. And according to the number of the holes made, calculating
Premix Ex Taq II (2X) and primer content were mixed in appropriate amounts to prepare a Premix. Premix was added to the bottom of the corresponding PCR tube, 5.8. mu.l/tube. The diluted DNA template was then added to the middle wall of the corresponding PCR tube, 4.2. mu.l/tube. When the same DNA template is added, the gun tip does not need to be replaced.
Control group Real timePCR reaction (10. mu.l): according to the number of holes made, calculate
Premix Ex Taq II (2 ×), primers, and sterile distilled water content. Mixing the three materials according to the corresponding amount to prepare a premixed solution. The premix was added to the bottom of the corresponding PCR tube at 9. mu.l/tube. Then DNA template was added to the middle wall of the corresponding PCR tube at 1. mu.l/tube. When the same DNA template is added, the gun tip does not need to be replaced.
The DNA content of the experimental group and the control group is the same, and only the sample volume is different.
After sample addition, the PCR premix solution and the template are fully mixed by using an eight-tube centrifuge for instantaneous centrifugation.
2.2 fluorescent quantitative PCR detection.
Performing fluorescent quantitative PCR amplification. Standard procedure for PCR amplification: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 20s, 40 cycles. Dissolution curve: 95 ℃ for 10s, 65 ℃ for 60s and 97 ℃ for 1 s.
2.3 repeated precision determination of different template loading volumes.
The assay was repeated 6 times at different loading volumes. From the measured Ct values, the coefficient of variation (CV%) of the Ct values was calculated and compared.
2.4 intermediate precision measurement of sample volumes of different templates.
The experiments were performed 3 times at different times, and 3 replicates were performed at each time. From the measured Ct values, the coefficient of variation (CV%) of the Ct values was calculated and compared.
3. Results
3.1 repeated precision measurements at different sample application positions.
After 6 repeated detections, comparing the repeated precision of the methods corresponding to the traditional sample adding position and the sample adding position of the invention, the CV (%) of the Ct value is less than 3%, which shows that 2 sample adding positions have good repeated precision (table 1).
TABLE 1 repeated precision determination of different sample application positions
3.2 measurement of intermediate precision at different sample application positions.
When 3 independent repeated tests were performed at different times, and the intermediate precision of the traditional sample loading position and the sample loading position of the present invention were compared, CV (%) of Ct values were all < 3%, indicating that all the intermediate precision was good (table 2).
TABLE 2 measurement of intermediate precision at different sample application positions
During the application of sample, although the DNA template adds middle part pipe wall and tube bottom and all has better precision, nevertheless can not contact PCR premix liquid when adding the middle part pipe wall, can not lead to the rifle point to pollute, consequently need not change the rifle point, labour saving and time saving saves the rifle point.
3.3 repeated precision determination of sample volumes for different templates.
After 6 repeated detections, the repeated precision of different template sample adding volumes is compared, and compared with the traditional template sample adding volume (1 μ l), the CV (%) of the Ct value of the template sample adding volume (4.2 μ l) method is smaller and less than 3%, and good repeated precision is shown (Table 3).
TABLE 3 repeated precision determination of different template loading volumes
3.4 intermediate precision measurement of sample volumes of different templates.
The independent repeated experiments were performed 3 times at different times, and the intermediate precision of different template loading volumes was compared, and the CV (%) of the Ct value of the template loading volume (4.2 μ l) method of the present invention was smaller and < 3% compared to the conventional template loading volume (1 μ l) method, showing good intermediate precision (table 4).
TABLE 4 intermediate precision determination of different template loading volumes
In conclusion, the sample adding position of the DNA template does not influence the precision of the real-time fluorescence PCR, but the gun tip does not need to be replaced when the DNA template is added to the middle tube wall (the template is the same), so that time and labor are saved, and the gun tip is saved; the sample adding volume of the DNA template is firstly enlarged and then added into the PCR reaction system, so that the precision of the real-time fluorescence PCR detection can be improved and the error of the real-time fluorescence PCR detection can be reduced in the low-volume PCR reaction system. The sample adding method combines the sample adding position and the sample adding volume, saves more time and labor, saves reagents and gun tips and has higher precision compared with the traditional sample adding method.
Claims (8)
1. A sample adding method of a PCR reaction system is characterized in that: the method comprises the following steps:
1) adding the PCR premix solution to the bottom of a PCR tube;
2) diluting the DNA template with water;
3) and (3) adding all the diluted DNA templates into a PCR tube, and centrifugally mixing to obtain a PCR reaction system.
2. The method of claim 1, wherein: in step 3), adding the DNA template into the middle tube wall of the PCR tube.
3. The method of claim 1 or 2, wherein: in the step 2), the dilution multiple is 3-6 times.
4. The method of claim 3, wherein in step 2), the dilution factor is 4.2.
5. The method of claim 1 or 2, wherein: the total volume of the PCR reaction system is 10-25 mu l.
6. The method of claim 5, wherein: the total volume of the PCR reaction system is 10 mu l.
7. A PCR method, characterized by: the PCR method is used for preparing the PCR reaction system by using the sample adding method of the PCR reaction system of any one of claims 1 to 6.
8. The PCR method of claim 7, wherein: the PCR is real-time fluorescent quantitative PCR.
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|---|---|---|---|---|
| CN102296117A (en) * | 2011-09-06 | 2011-12-28 | 北京大北农科技集团股份有限公司 | Rapid qualification and quantitation measurement method of saccharomyces cerevisiae in additive premix sample |
| CN111057751A (en) * | 2020-01-19 | 2020-04-24 | 南京林业大学 | Method for detecting number of soil fungi based on real-time fluorescent quantitative PCR |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296117A (en) * | 2011-09-06 | 2011-12-28 | 北京大北农科技集团股份有限公司 | Rapid qualification and quantitation measurement method of saccharomyces cerevisiae in additive premix sample |
| CN111057751A (en) * | 2020-01-19 | 2020-04-24 | 南京林业大学 | Method for detecting number of soil fungi based on real-time fluorescent quantitative PCR |
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| 林先武: "棉铃虫蛹滞育的糖代谢相关基因研究" * |
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Application publication date: 20211231 |