Summary
Ring-mediated thermostatic amplification refers to the technique of nucleic acid amplification in a short period of time by using four specific primers at 60~65 ℃ isothermal environment, where the DNA is in dynamic equilibrium, and relying on a high-activity strand to displace the DNA polymerase.
The reaction produces a large amount of amplification product, i.e., a white precipitate of magnesium pyrophosphate, and the presence or absence of the white precipitate can be observed with the naked eye to determine the presence or absence of the target gene.
Operation method
LAMP (loop-mediated isothermal amplification)
Materials and Instruments
Materials: plasmid extraction kit, primers, DNA polymerase, dNTPs Mix, genomic DNA; Move 1. Primer design The target gene was selected according to the National Center for Biotechnology Information (NCBI) (nih.gov), sequence comparison was carried out, and the conserved region was selected according to the sequence comparison results, and the LAMP primers were designed using the online software, Primer, and synthesized by Beijing Kinko Biotechnology Co.
Instruments: Gradient PCR, turbidimeter, etc.
Amplify the target bands and ligate them to the suitable vector, obtain the target plasmid by monoclonal picking and shaking, and extract the plasmid according to the instructions.
3. LAMP reaction system and its optimization1) LAMP reaction system
0.4 μL of each internal primer (100 μmol/L), 0.5 μL of each external primer (10 μmol/L), 1 μL of DNA polymerase, 1 μL of template, 1.4 μL of dNTPs (25 mmol/L), 1.5 μL of Mg2+ (100 mmol/L), and 2.5 μL of 10 × Thermopol Buffer, replenished to 25 μL with ddH2O. The solution was made up to 25 μL with ddH2O.
(2) Optimization of LAMP reaction system
The LAMP reaction temperature was set at 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃ and 65 ℃ to determine the optimal reaction temperature of the system; the reaction time was set at 15~60 min/group, with a total of 10 groups and an interval of 5 min between each group; the final concentration gradient of Mg2+ was set at 2, 4, 8, 12, and 16 mmol/L, which meant that the final concentration of Mg2+ was increased by adding 100 mmol/L of Mg2+ stock solution 0.5 mgol/L.
The final concentration gradient of Mg2+ was set at 2, 4, 8, 12 and 16 mmol/L, i.e., 100 mmol/L of Mg2+ storage solution was added 0.5, 1, 2, 3 and 4 μL respectively; the final concentration ratios of different internal and external primers were set at 2:1, 4:1, 8:1, 16:1 and 32:1 (0.5 μL of external primer was added fixedly); and the amount of DNA polymerase added was set at 4, 8, 16 and 32 U (corresponding to the addition of 0.5, 1, 2, 3 and 4 μL of DNA polymerase, respectively).
At the end of the reaction, 2.5 μL of the sample and 0.5 μL of 6× DNA sampling buffer were mixed and separated by 2% agarose gel electrophoresis to determine the test results, with the positive results showing a characteristic stepped band, and the negative results showing no such characteristic band.
(3) Specificity and sensitivity of LAMP detection of target gene fragments
Genomic DNA was used as a template (1 μL) for specificity testing. At the same time, the plasmid containing the target gene was used as a positive control, and the reaction was carried out at the optimized reaction temperature and reaction time, and the results were detected by agarose gel electrophoresis after the completion of the reaction.
Sensitivity test: Dilute the plasmid containing the target gene with ddH2O to 5, 10, 50, 102, 103, 104, 105copies/μL, and take 1 μL as the template to perform the reaction at the optimized reaction temperature and reaction time, and then test the results by agarose gel electrophoresis after the completion of the reaction.
4. Extension reactionAfter screening the corresponding reaction system, denaturing and annealing, the reaction system (except the enzyme) was heated at 95 ℃ for 5 min, then cooled, and then added with DNA polymerase; isothermal extension, holding and extending for a certain period of time under the screened time; and termination of the reaction at a temperature higher than 80 ℃ for 2 min.
5. Detecting LAMP product by observing the amount of white precipitateAs the LAMP reaction proceeds, dNTP releases pyrophosphate ions, producing a white precipitate of magnesium pyrophosphate. The amount of white precipitate produced is proportional to the amount of double-stranded DNA in the reaction solution, so the amount of white precipitate can be measured by turbidimetry to characterize the extent of the reaction. The precipitate can only be observed with the naked eye when the amount of dNTP in the reaction system reaches 0.5 M. The amount of dNTP in the reaction solution is proportional to the amount of double-stranded DNA in the reaction solution.
Common Problems
1, LAMP amplification is a chain replacement synthesis, the length of the gene should be within 300 bp. 2、LAMP is highly specific and easily contaminated to produce false positives, so care should be taken to prevent contamination during the experiment.
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