Primer design key factors and design skills
It is better to synthesize the primers. In addition to the difference between the Tm of the pre-primer and the post-primer, we must also consider the following factors:
First, the GC content
The GC content of the primer is generally 40-60%, preferably 45-55%, and too high or too low is not conducive to the initiation of the reaction. Some GCs have low or high GC content, which makes the GC content of the primers not within the above range. At this time, the GC content and Tm value of the upstream and downstream primers should be kept close to each other (the GC content of the upstream and downstream primers cannot be too different) Large) to favor the choice of annealing temperature. If the GC ratio is exceeded, add As or Ts at the 5' end of the primer; if the AT ratio is too high, then increase Gs or Cs at the 5' end. However, some people think that it is generally wrong to think that PCR primers should have a GC/AT ratio of 50%. Using human genomic DNA as a template, 81% AT primers can produce a single, specific, 250 bp long. , product containing 70% AT. There is no need to calculate the melting temperature of the product and the primer at all, and the GC/AT ratio of the PCR primer should be equal to or higher than the GC/AT ratio of the template to be amplified. The GC content of the product is preferably greater than the GC content of the primer.
Second, Degeneracy polysemy
When designing ambiguous primers, primer ambiguity should be minimized, which leads to better specificity. The ambiguity of the 3 ends should be avoided as possible, because even a single base mismatch can prevent primer extension.
Third, 3' End Stability 3 end stability
Primer stability affects its mismatch efficiency. An ideal primer should have a more stable 5-terminal end and a relatively stable 3 end. If primer 3 is highly stable, it is possible to cause mismatches even if the 5 ends are not paired, resulting in non-specific amplification bands. The reason why primers with low stability at the 3 terminus are better is that when the primers are mismatched, it is difficult to extend due to unstable binding of the primers which are less stable at the 3 terminus.
Fourth, GC Clamp GC clamp
The efficient binding of the primer to the site of interest requires a stable 5 terminus. The 5 ends with strong stability in this section are called GC clamps. It ensures a stable binding of the primer to the template. Selection of primers with suitable stability minimizes the annealing temperature while ensuring that non-specific bands are not produced.
Five, Secondary Structures secondary structure
Secondary structure is an important factor that must be considered in primer design. The secondary structure can significantly affect the number of primers that can correctly bind to the template in the reaction. The presence of the hairpin structure can limit the binding ability of the primer to the target site, thereby reducing the amplification efficiency, and the primers forming the hairpin loop cannot be in the PCR amplification. Play a role.
Six, Hairpin card issuance structure
The formation of the hairpin structure is due to the secondary structure formed by the folding of the primers in the complementary base of the primer itself, and since the formation of the hairpin structure is an intramolecular reaction, only three consecutive base pairings are required. The stability of the hairpin structure can be measured by free energy. The free energy size depends on the energy released by the base pairing and the energy required to fold the DNA to form the hairpin loop. If the free energy value is greater than 0, the structure is unstable and does not interfere with the reaction. If the free energy value is less than 0, the structure can interfere. reaction.
Seven, Dimer dimer
The paired regions between the primers are capable of forming a primer dimer which is a secondary structure formed between the same or different two primers. It causes amplification of the primer dimer and reduces the amplification product of interest. The dimer can be formed between two primers with the same sequence or between the forward and reverse primers. If the paired region is more serious at the 3 terminus, the 3 ends are paired. Primer dimer amplification is easily caused.
Eight, False Priming mismatch
If the primers can bind to other regions than the site of interest, the amplification efficiency will significantly reduce the target product band to be reduced or smear. 3 The tendency of a few base pairs to form a mismatch at the end is higher than the same number of base pairs in the upstream region of the primer. When using the primer design software, you can set the 3 ends of the mismatch or the full length of the primer. The number of consecutive base pairs.
Nine, the issue of free energy (how to judge the quality of the primer based on free energy)
1. The ΔG value (free energy) reflects the strength of the combination of the primer and the template. In general, the ΔG value of the primer is preferably sinusoidal, that is, the 5' end and the intermediate ΔG value are higher, and the 3' end ΔG value is relatively lower, and should not exceed 9 (the ΔG value is negative, here is absolutely Value), this will help to properly initiate the reaction and prevent false triggers. When the ΔG of the 3′-end double-strand is 0～-2 kcal/mol, the PCR yield is almost 100%, and the yield gradually decreases with the increase of its absolute value, only 40% at -6, less than 20% at -8. And -10 is close to 0.
2. The energy of the primer dimer and hairpin structure should not exceed 4.5. Otherwise, the primer dimer band will be easily generated and the concentration of the primer will be lowered, resulting in the failure of the normal PCR reaction. One parameter related to the dimer is the base. Distribution, continuous GGG or CCC at the 3' end will cause an error to be triggered. The higher the energy value of the dimer formation, the more stable and the less satisfactory. As with the dimer, the lower the energy value of the hairpin structure, the better. Although some have a hairpin loop, a self-complementary primer with a ΔG of -3 kcal/mol can give good results, but if its 3' end is occupied by a hairpin loop, it is troublesome, that is, it will cause the inside of the primer. The extension reaction reduces the number of primers involved in the formal reaction. Of course, if the hairpin loop reacts at the 5' end, it does not have much effect.
X. Design problems of PCR primers for products to be sequenced
In DNA sequencing PCR, it is best to use a primer that is stable at the 5' end (such as a high GC content) and a 3' end is not stable (such as a high AT content). The structure of this primer can effectively eliminate the pseudo-priming reaction. . This is the experience based on the internal stability of the primers. The reason why primers with low stability at the 3' end can play a good role in these reactions is that the degree of stability of the base formed near or at the 3' end with the base of the non-target site is not sufficient to trigger DNA. Synthetic, so no fake products are produced. Therefore, in order to efficiently initiate the reaction, the 5' end and the central portion of the primer must also form a double strand with the target DNA. In contrast, an oligonucleotide with a stable, GC-rich 3' end does not require all of its nucleotide sequences to be paired with the target sequence, only by virtue of its 3' end to a strong fit to any position of the target sequence. The reaction can be initiated to produce a non-specific product. In any event, the stability of the last 5 nucleotides at the 3' end of the oligonucleotide is less than -9 kcal/mol, which is usually a specific probe or primer. The more unstable the 3' end of the oligonucleotide, the lower the probability of false initiation.
By the way, if the novice is just starting to touch the primer design, Primer Premier 5.0 is recommended because it is simple and easy to learn and use; if you want to design the primers to perfection, the recognized preferred software is Oligo, and secondly I think it is DNAstar. Oligo is powerful, so it's not as easy to use as Primer Premier 5.0. First design with Primer Premier 5.0, then take the designed primers into Oligo to test the pros and cons of this pair of primers, I think this is a good choice for most primer designers!
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