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In the previous issue, we introduced the development history of PCR and the basic technical principles of qPCR. In this issue, we will also start to explain the primer design of qPCR experiment as promised!
As a branch of PCR technology, the primer design of qPCR also follows the basic rules of PCR primer design. At the same time, since the quantitative methods of qPCR are mainly divided into dye method and probe method, there are also differences in primer design. The rules followed by the SYBR dye method in primer design are not much different from those of conventional PCR. The following points should be noted: 1. The primer length is generally between 17 and 25 bases, and the G+C content is between 40% and 60%; 2. The last base at the 3' end of the primer has a great influence on the efficiency of Taq enzyme DNA synthesis. Different last bases at the mismatch position lead to different amplification efficiencies. The mismatch rate of the last base A is significantly higher than that of the other three bases, followed by T. Therefore, the use of bases A or T at the 3' end of the primer should be avoided. In addition, primer dimers or hairpin structures may also cause PCR reaction failure. The 5' end sequence has little effect on PCR, so it is often used to introduce modification sites or markers; 3. The primer bases should be randomly distributed, and they cannot have four consecutive bases that are complementary to each other. The primers cannot have four consecutive bases that are complementary to each other. 4. The Tm value of the template position sequence corresponding to the primer is around 72℃, which can make the annealing condition optimal. There are many ways to calculate the Tm value, such as according to the formula Tm＝4（G＋C）＋2（A＋T）. The nearest neighbor method is used in Oligo software. Try to use the same Tm value for the two primers (preferably the difference should not exceed 5℃). The annealing temperature is selected according to the lower Tm value. The annealing temperature of the two primers can also be balanced by changing the length of the primers. For longer primers, the Tm value needs to consider the kinetic parameters and is obtained from the "nearest neighbor" calculation method. Most of the existing PCR primer design software adopts this method; 5. ΔG value refers to the free energy required for the formation of a double-stranded DNA, which reflects the relative stability of the base pairs within the double-stranded structure. Primers with a low ΔG value at the 3' end (the absolute value does not exceed 9) and relatively high ΔG values at the 5' end and in the middle should be selected. If the ΔG value at the 3' end of the primer is too high, it is easy to form a double-stranded structure at the mismatch point and trigger a DNA polymerization reaction; 6. The energy value of primer dimers and hairpin structures is too high (more than 4.5 kcal/mol), which can easily lead to the formation of primer dimers and reduce the effective concentration of primers, making the PCR reaction unable to proceed normally. The product cannot form a secondary structure; 7. The 5′ end of the primer can be modified, but the 3′ end of the primer cannot be modified; 8. The quantitative product is around 100-300; 9. It is best to design primers that span an intron. When designing primers for the TaqMan probe method, in addition to all the principles of the SYBR dye method, due to the existence of the probe, extra attention should be paid to the 5' end without a G base, because the G base will quench the fluorescence emitted by the luminescent group. In addition, it is necessary to ensure that the TaqMan probe annealing temperature is 10°C higher than the primer, there are no consecutive identical bases, the probe position is as close to the upstream primer as possible, and the C base is far more than the G base. Considering that the current work of designing primers mainly relies on professional software on PC, I will not explain more about the precautions when designing here. By the way, I would like to recommend several commonly used primer design software/websites: software ：Primer Premier, Oligo7, Beacon Designer Website : Primer-blast (NCBI), Primer3 Plus, Primerbank, BiSearch, Pubmed |
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If it is right RNA If the sample is to be quantified, the following points should be considered when designing primers:
1 , primer specificity Many people think that primer specificity refers to the singleness of the amplified product. In fact, the interference of similar sequences in the transcriptome is often the main cause of false positive detection, especially when the expression level of the target transcript is low and the expression level of similar sequences is high. For the detection of the singleness of the amplified product, it is necessary to check whether the melting curve is single and whether the electrophoresis diagram of the PCR product is single. If you need to ensure high singleness, you can also use Labchip for product analysis (which can distinguish fragments of 4bp in size). 2 , variable shear In addition, special attention should be paid to variable splicing. Variable splicing of RNA can lead to the production of different transcripts and result in translation encoding the formation of multiple proteins. Variable splicing is an important mechanism for regulating gene expression and producing protein diversity. It is estimated that the probability of variable splicing in the human genome is as high as 60%. When it is necessary to detect the transcription level of a specific spliceosome, the primer design should limit the amplicon to a segment that is different from other spliceosomes. Otherwise, the detected transcription level is the sum of all spliceosomes rather than a specific spliceosome. 3 , amplification efficiency If the ΔΔCt method is used for the final calculation, the target gene and the reference gene need to have similar amplification efficiencies. The best primer amplification efficiency should be between 90% and 110%, and the linear regression coefficient R 2 Greater than 0.98. 4 , Primer production process During the production process of primers, there is generally a 0.01% error rate (depending on the manufacturer, there will be slight differences). In addition, there are byproducts (desalting purification) and a large amount of salt (especially page purification). Although this does not necessarily affect the PCR experiment, it may cause experimental instability. Generally, primers and probes used in clinical reagents will try to avoid this problem, while most scientific research primers often ignore this problem. 5 , whether the primer spans an intron During the nucleic acid extraction process, the extracted RNA often contains a small amount of DNA (the proportion varies depending on the extraction kit and the operation itself), which will also affect the experimental results. Generally, primers that span introns can effectively distinguish RNA and DNA samples. Only gene expression in RNA can be detected. |
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6 , primer design close to 5 ' End or 3 ' end Generally speaking, influenced by the differences in reverse transcription efficiency and mRNA degradation preference, primers close to the 3' end can better reflect gene expression.
Below we will share the primer design process with you using NCBI online primer design as an example: |
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1. Search for the target gene we want on NCBI. Click Pick Primers in the menu bar on the right as shown below: |
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2. After that, you will enter this screen and set the parameters according to our needs, as shown below : |
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3 . We click Get primers After that, we may get several pairs of primers, as shown below: |
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4. We can just select the best primer pair we want from these primer pairs, isn’t it so easy? |
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5. Finally, we can verify the uniqueness of the primer by Primer-BLAST Enter our primer sequences for verification: |
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6. After verification, you can send it to the company for synthesis. Of course, we still need to do the last step, which is the PCR verification of primers, especially the verification of product specificity. Often, the primers we design perform perfectly in the software, but in actual PCR, there will be problems with non-specific amplification. This may be due to the primers themselves, or the reagents, the system, salt ion concentration, etc. Good reagents are helpful assistants in solving non-specific amplification. After N rounds of formula optimization, Starligter SYBR qPCR Mix can help you solve the problem of non-specific amplification, simplify the process of primer design and verification, and save your time and cost! |
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Our Products Qihengxing new product launch | StarLighter SYBR Green qPCR Mix !
Come on guys, design your own exclusive primers! If you have any questions, please scan the QR code below and communicate with our technical teachers! |
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The trial activity we released last time was very effective. With the approval of the leaders, the trial activity will continue! Participation method: Scan the QR code below to participate in our trial gift. As long as you try our related products and give feedback on the trial results, we will send gifts to you. The number of gifts is limited, so sign up quickly to get the gifts! |
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Ps（ Leaders don't let me say ): We will send out benefits through the WeChat platform every week in the future, making a series of push notifications, including: Experimental design , Nucleic acid extraction and quality control , Reverse Transcription Solutions , Data analysis The theme of this series; the theme of the next issue is Experimental design , stay tuned. |
