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In the previous issue, we introduced the history of PCR and the basic technical principles of qPCR. In this issue, we will continue our discussion of primer design for qPCR experiments!
As a branch of PCR technology, qPCR primer design also follows the basic rules of PCR primer design. At the same time, because qPCR quantitative methods are mainly divided into dye method and probe method, there are also differences in primer design. The rules followed in primer design for the SYBR dye method are not much different from those for conventional PCR. The main points to note are: 1. Primer length is generally between 17 and 25 bases, with a G+C content between 40% and 60%; 2. The last base at the 3' end of the primer has a significant impact on the efficiency of Taq enzyme DNA synthesis. Different last bases lead to different amplification efficiencies at the mismatch position. 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 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. A Tm value of around 72°C for the primer sequence at the template position provides optimal annealing conditions. There are various methods for calculating Tm values, such as the formula Tm = 4(G + C) + 2(A + T). Oligo software uses the nearest neighbor method. Whenever possible, use the same Tm value for both primers (ideally, a difference of no more than 5°C). The annealing temperature is selected based on the lower Tm value. Alternatively, the annealing temperatures of the two primers can be balanced by varying the primer length. For longer primers, the Tm value requires consideration of kinetic parameters and is calculated using the "nearest neighbor" method. Most existing PCR primer design software utilizes this method. 5. The ΔG value refers to the free energy required for DNA duplex formation and reflects the relative stability of base pairs within the duplex structure. Primers with a low ΔG value at the 3' end (absolute value no more than 9) and relatively high ΔG values at the 5' end and in the middle should be selected. A primer with a ΔG value that is too high at the 3' end of the primer can easily form a duplex structure at the mismatch point and trigger DNA polymerization. 6. The energy value of primer dimers and hairpin structures is too high (over 4.5kcal/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 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 incorporating all the same principles as the SYBR dye method, due to the presence of the probe, extra care must be taken to avoid a G base at the 5' end, as this quenches the fluorescence emitted by the luminescent group. Furthermore, it is important to ensure that the TaqMan probe annealing temperature is 10°C higher than the primer, that there are no consecutive identical bases, that the probe is positioned as close as possible to the upstream primer, and that C bases significantly outnumber G bases. Considering that the current work of primer design 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 RNA If the sample is to be quantified, the following points should be considered when designing primers:
1 , primer specificity Many people assume that primer specificity simply means a single amplified product. However, interference from similar transcriptome sequences is often a major cause of false positives, especially when the target transcript is expressed at low levels while similar sequences are expressed at high levels. To determine the uniqueness of amplified products, it's necessary to examine the uniqueness of the melting curve and the electrophoresis pattern of the PCR products. If high uniformity is required, product analysis using Labchip (which can distinguish fragments as small as 4 bp) is also recommended. 2 , variable shear In addition, special attention should be paid to alternative splicing. Alternative splicing of RNA can result in the production of different transcripts, which in turn lead to the translation and encoding of multiple proteins. Alternative splicing is an important mechanism for regulating gene expression and generating protein diversity. It is estimated that the probability of alternative splicing in the human genome is as high as 60%. When detecting the transcript level of a specific spliceosome, primer design should limit the amplicon to a region that is distinct from other spliceosomes. Otherwise, the transcript level detected 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 primer production process, there's typically a 0.01% chance of error (this rate varies slightly depending on the manufacturer). Furthermore, there are side products (from desalting and purification) and large amounts of salt (especially during PAGE purification). While these factors don't necessarily affect PCR experiments, they can lead to experimental instability. While primers and probes used in clinical reagents generally minimize this issue, most research primers often overlook it. 5 , whether the primer spans an intron During nucleic acid extraction, the extracted RNA often contains a small amount of DNA (the ratio varies depending on the extraction kit and the procedure itself), which can affect experimental results. Generally, intron-spanning primers can effectively distinguish between RNA and DNA samples, allowing detection of gene expression only in RNA. |
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6 , primer design close to 5 ' End or 3 ' end Generally speaking, due to differences in reverse transcription efficiency and mRNA degradation preferences, 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 select the best primer pair we want from these primer pairs. That’s it, isn’t it? |
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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 complete the final step, which is the PCR verification of the primers, especially the verification of product specificity. Often, the primers we design perform perfectly in the software, but non-specific amplification problems may occur during actual PCR. This may be due to the primers themselves, or to the reagents, such as the system, salt ion concentration, etc. Good reagents are helpful in solving non-specific amplification problems. Qihengxing Starligter SYBR qPCR Mix has undergone multiple rounds of formulation optimization to help you effectively solve the problem of non-specific amplification, simplifying the primer design and verification process for you, saving you time and costs! |
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Our Products Qihengxing new product launch | StarLighter SYBR Green qPCR Mix !
Come and 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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Our last trial was very successful, and with leadership's approval, we're continuing! To participate: Scan the QR code below to participate in our trial gift program. As long as you try our products and provide feedback on your experience, we'll give you a gift. Quantities are limited, so sign up quickly to get your hands on some! |
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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. |
