Step 1: In the process of finding and comparing the target gene in the first step, we can use the NCBI genbank sequence and DNAstar software to find and compare the target DNA or RNA-which I believe many people have done when analyzing the sequencing report. In this step, it is important to ensure that the analyzed sequence is in an overlapping group (overlapping group, that is, the overlapping of adjacent DNA fragments in some regions of chromosomes).
Step 2: If other conditions are the same, then the second step-the design of primer probe can be said to be the key to the success or failure of quantitative PCR. Summarize the following basic principles through various experiences:
Firstly, the probe is selected, and then the primer is designed to be as close to the probe as possible.
The selected sequence should be highly specific, and the amplification fragment with the smallest secondary structure should be selected as far as possible, because the secondary structure will affect the reaction efficiency and hinder the amplification of the enzyme. It is suggested to detect the secondary structure first. If the secondary structure is unavoidable, the annealing temperature should be increased accordingly.
The amplification length should not exceed 400bp, and ideally it should be between 100- 150bp. The shorter the amplification fragment, the easier it is to obtain an effective amplification reaction. Shorter amplified fragments can also easily ensure the consistency of analysis.
When the GC content is kept between 20% and 80%, non-specific reaction will easily occur in the GC enrichment area, which will lead to the reduction of amplification efficiency and non-specific signals in fluorescent dye analysis.
In order to ensure efficiency and repeatability, we should avoid repeating nucleotide sequences, especially G (there cannot be four consecutive G's).
Primers and probes are paired with each other to avoid the formation of dimer and hairpin structure.
Sequence selection should be in the conservative part of the gene
Avoid the formation of four or more consecutive pairs between primers themselves or between primers, and avoid the formation of ring hairpin structure by primers themselves.
Typical primers 18 to 24 nucleotides long. Primers need to be long enough to ensure the uniqueness of the sequence and reduce the possibility that the sequence exists at non-target sequence sites. However, primers longer than 24 nucleotides do not mean higher specificity. Longer sequences may hybridize with mismatched sequences, which reduces specificity and is slower than short sequences, thus reducing yield.
Tm value is 55-65℃ (because exonuclease activity of nucleic acid is the highest at 60℃), and GC content is 40%-60%.
The TM difference between primers should be avoided to exceed 2℃
Base A is avoided at the 3' end of the primer, and three or more consecutive identical bases are avoided at the 3' end of the primer.
In order to avoid genome amplification, it is best to design primers spanning two exons.
Taqman probe technology requires the fragment length to be 50bp- 150bp.
The primer end (last 5 nucleotides) cannot exceed 2 G and C.
The probe is as close as possible to the upstream primer.
The probe length should be 15-45bp (preferably 20-30bp) to ensure the binding specificity.
DNA folding and secondary structure of detection probe
Tm value is 65-70℃, which is usually 5- 10℃ higher than that of primer (at least 5℃), and GC content is 40%-70%.
G guanine should be avoided at the 5' end of the probe-because 5'G will have quenching effect, even if it is cut off, it will still have quenching effect.
In the whole probe, the content of base C is obviously higher than that of G-G, which will reduce the reaction efficiency, so another strand should be selected as the probe.
In order to ensure the specificity of primer probe, it is best to verify the designed sequence once in blast. If there are nonspecific complementary regions, it is suggested to redesign the primer probe.
G is avoided at the 5' end of the probe. Even if the probe is hydrolyzed into a single base, the G base linked to the reporter group can still quench the fluorescence signal of the group.
Tm value should be 65-67℃.
Try to shorten the Taqman MGB probe, but the probe length should not be less than 13bp.
Try to avoid repeating bases, especially G bases, and avoid four or more G repeats.
In principle, as long as there is a base mutation in the MGB probe, it will be detected by the MGB probe (the MGB probe will not hybridize with the target fragment and will not generate a fluorescence signal). Therefore, in SNP detection, in order to detect mutation, that is, Taqman MGB does not hybridize with the target fragment and does not produce fluorescence signal, the mutation site of the probe should be placed in the middle of 1/3 as much as possible.
Note: In order to meet the above four requirements, the mutation site of the probe can be moved to the 3' end, but the mutation site is at least 2 bases away from the 3' end (that is, the last two bases of the probe must be absolutely conserved) for SNP detection. On the other hand, if the same kind of detection is to be carried out, it is necessary to find a conservative fragment region, and there can be no mutation sites in the probe. Even if the probe is only 13 bp, the probe is still not completely conserved. There are several mutations, and the mutation site should be close to the 5' end of the probe, so that even if there are mutations, the probe can hybridize with the target fragment and generate fluorescence signals. Another method is to design degenerate probes, even mutations can be detected.
Step 3: Find a trustworthy company to synthesize primers and probes. Generally, primer synthesis is familiar to everyone, and the price is relatively cheap (especially in the last two years), while probes are more expensive. Generally, the synthesis of Taqman probes ranges from 65,438+0,000 yuan to 5,000 yuan (different synthesis requirements have different prices)-and this is only the price tag, and the sequence synthesis is basically the same as primer synthesis.
Step 4, Step 5 and Step 6: The general quantitative PCR reaction system is not much different from the general PCR except that Taqman probes are added. The other difference is the step-by-step method. It should be pointed out that:
The amplification enzyme is preferably a hot start enzyme.
It is necessary to optimize the concentration of primers and probes. It is recommended to optimize the concentration from 50nM to 900 nm, generally 200nM (note that the probe needs to be stored away from light.
Similarly, the dosage of Mg+ and enzyme need to be optimized, and the recommended reaction concentration of enzyme is 1.25- 1.5U(50ul).
The addition amount of DNA template is usually below 100 ng. Because different kinds of DNA templates contain different copies of the target gene, gradient dilution can be carried out when necessary to determine the optimal addition amount of DNA templates. If the second PCR amplification reaction of two-step RT-PCR reaction is to be carried out, the amount of RT reaction solution used as DNA template in the first step should not exceed 10% of the total volume of PCR reaction solution.
In addition, although the cycle parameters are determined after the design of primers and probes, they sometimes need to be optimized.
Step 7: In data analysis, standard curves are usually generated from Ct values of standard samples with different concentrations, and then the related equations are calculated. Tilt equation
Degree can be used to check the efficiency of PCR. For the PCR efficiency of 100%, the ideal slope is 3.32. The best standard curve is that the amplification efficiency based on PCR is 90%- 100 %( 100% means that every cycle, the total number of templates will be doubled). The linear regression analysis of all standard curves requires a high correlation coefficient (R2≥0.99), so that the experimental process and data can be considered credible. Using this equation, we can calculate the initial template quantity of unknown samples. Most quantitative PCR instruments have such a software, which can automatically calculate the initial template amount of unknown samples from the standard curve.
There are two basic methods: absolute quantification and relative quantification, which researchers need to choose according to their own experimental purposes. Absolute quantification refers to comparing unknown samples with standard curves for analysis. A common standard is a DNA sample with a known absolute concentration. It should be noted that the accuracy of absolute quantification is relative to the accuracy of standard. Relative quantification means that two or more genes are compared with each other, and the result is a ratio, and the exact number is not detected.
In addition, because there are some differences between different samples in the reaction process, in addition to making standard curves for quantification, it is necessary to design internal reference genes with relatively stable expression levels to standardize the results. β -actin and glyceraldehyde triphosphate dehydrogenase (GAPDH) are two commonly used housekeeping genes, as well as cyclophilin, 18sr RNA, phosphoinositide kinase, β-microglobulin, β-glucosidase, hypoxanthine ribotransferase and transfer receptor. It should be noted that these genes will be affected by reaction conditions. When designing quantitative expression research, it is necessary to ensure the quality of initial control genes. Strict researchers will standardize quantitative data by taking the geometric average of quantitative results of a series of internal reference genes.
Another problem is how to judge the quality of the obtained data. As for the amplification curve, the empirical conclusion is:
1. Overall, the inflection point of the curve is clear, the exponential period is obvious, the overall parallelism of the amplification curve is excellent, the baseline is flat and there is no upward phenomenon, and the exponential period of the amplification curve of low concentration samples is obvious.
Mg2+ concentration is the key factor affecting Taq enzyme activity. Too low Mg2+ concentration will affect the optimal activity of Taq enzyme, while too high Mg2+ concentration will increase nonspecific amplification.
Generally speaking, the concentration of Mg2+ should be 2 ~ 5 mmol/L for real-time qPCR reaction with DNA or cDNA as template, and 4 ~ 8 mmol/L for direct reaction with mRNA as template.
Template concentration should be selected according to Ct value. Generally speaking, it is appropriate to make the Ct value between 15 ~ 30 weeks. If it is greater than 30, a higher template concentration should be used. If the Ct value is less than 15, the template concentration should be reduced. If the researcher is conducting the experiment for the first time, he should choose a series of templates with diluted concentrations to conduct the experiment to select the most suitable template concentration.
It is used for real-time qPCR reaction of some samples with complex components (soil, sediment, etc.). ), the extracted DNA template usually contains more humus and other substances, which will inhibit the PCR reaction and lead to the decrease of amplification efficiency, thus reducing the quantitative accuracy of the target gene.
Usually, DNA templates are diluted to eliminate PCR inhibition, but in some cases, due to the small amount of templates, the dilution method is not suitable, so additional purification of templates is needed.
Too low primer concentration will lead to incomplete reaction. If the primer concentration is too high, mismatch will occur and non-specific amplification products will be produced. For most PCR reactions, 0.5μmol/L is the appropriate primer concentration. If the initial experimental results are not ideal, it can be adjusted between 0.3 ~1.0 μ mol/L. ..
The purity of primers used in real-time QCPR should reach at least PAGE level, and if OPC level primers are used, the results will be inaccurate.
Usually, the peak of the melting curve of the qPCR product of the target gene appears between 80℃ and 90℃. When the peak of low fluorescence intensity appears in the melting curve between 70℃ and 80℃, it indicates that primer dimer is produced in the real-time QCPR reaction.
At this time, the reason of primer dimer should be judged first. If the primer dimer does not appear in the high target gene copy sample, but only exists in the low target gene copy sample, then the primer dimer appears because the target gene content in the sample is too low, resulting in self-matching. We can eliminate primer dimer by increasing the amount of template or decreasing the amount of primer.
Another method to eliminate primer dimer is to increase the annealing temperature, but it is not necessarily effective. It is suggested that before real-time QCPR, it is best to detect whether the primer will form dimer by temperature gradient qPCR and determine the appropriate annealing temperature.
When drawing the standard curve of the target gene, the problem of large deviation of amplification efficiency is often encountered.
500μg/mL BSA can be added to the reaction system, which can stabilize the low concentration nucleic acid template and improve the amplification efficiency.
The amplification efficiency of short PCR products is higher than that of long PCR products, because short products are more easily denatured at 95℃, which makes primers and probes combine with their complementary sequences more effectively in the annealing stage, reducing the pollution caused by genome amplification and shortening the amplification time.
Increasing annealing temperature can improve the specificity and amplification efficiency of PCR reaction. If necessary, the annealing temperature can be increased to above 60℃, and the extension stage of 72℃ can be removed, so that annealing and extension can be combined to ensure the amplification efficiency of the target gene.
If the amplification efficiency of the standard curve is higher than 100%, it is mostly due to the operation error in the standard gradient dilution process, which leads to the inaccuracy of the low concentration standard with more dilution times. The standard curve should be redrawn after the standard is diluted again.
Reference: Biology