1. Primers are preferably designed in a conserved region of the template cDNA. Conserved regions of DNA sequences are determined by comparison of similar sequences between species. Searching for the same gene of different species on NCBI, through sequence analysis software (such as DNAman) alignment, the same sequence of each gene is a conserved region of the gene. 2. Primers are generally between 15 and 30 bases in length. Length of the primer (primer length) is commonly 18-27 bp, but not greater than 38, because it extends too long will cause a temperature greater than 74 deg.] C, the reaction does not apply to Taq DNA polymerase. 3. The GC content of the primer is between 40% and 60%, and the Tm value is preferably close to 72 °C. Too high or too low GC content is not conducive to the initiation of the reaction. The GC content of the upstream and downstream primers cannot differ too much . In addition, the Tm value of the upstream and downstream primers is the melting temperature of the oligonucleotide, that is, the temperature at which 50% of the oligonucleotides are double-stranded under a certain salt concentration . Effective starting temperature, generally higher than Tm value 5~10 °C. If the Tm value of the primer is estimated according to the formula Tm= 4(G+C)+2(A+T), the Tm of the effective primer is 55~80°C, and the Tm value is preferably close to 72°C to optimize the renaturation condition. . 4. The 3' end of the primer should avoid the third position of the codon. If the coding region is amplified, the 3' end of the primer should not stop at the third position of the codon, and the third position of the codon is prone to degeneracy, which will affect the specificity and efficiency of amplification . 5. The 3' end of the primer cannot select A, and it is best to choose T. When the 3' end of the primer is mismatched, there is a big difference in the efficiency of different base priming. When the base at the last position is A, even in the case of mismatch, the synthesis of the chain can be initiated. chain is T, mismatch initiation efficiency is greatly reduced, G, C mismatches between initiation efficiency a, between T, so the 3 'end of most good choice T. 6. Bases should be randomly distributed. The primer sequence should have no similarity in the template, especially the sequence with higher similarity at the 3' end, otherwise it will easily lead to error (False Priming). One way to reduce the similarity of the primer to the template is that the distribution of the four bases in the primer is preferably random, without the presence of polyfluorene or polypyrimidine . In particular, the 3' end should not exceed 3 consecutive G or C , as this would cause the primer to be erroneously triggered in the GC enrichment sequence region. 7. There should be no complementary sequences between the primer itself and the primer. The primer itself should not have a complementary sequence, otherwise the primer itself will fold into a hairpin structure (Hairpin) to renature the primer itself. This secondary structure affects the renaturation of the primer and the template due to steric hindrance. The primer itself cannot have a complement of 4 consecutive bases. Should not have complementarity between the two primers, in particular, the complementary overlapping 3 'ends should be avoided in order to prevent primer-dimer (Dimer and Cross dimer) is formed. There should be no four consecutive base complements between the primers. If the primer dimer and hairpin structure are unavoidable, try to keep the ΔG value too high (should be less than 4.5kcalmol). Otherwise, it is easy to cause a primer dimer band, and the effective concentration of the primer is lowered to prevent the PCR reaction from proceeding normally. 8. The 5' end and middle ΔG values ​​of the primer should be relatively high, while the 3' end ΔG value is lower. The ΔG value refers to the free energy required for DNA double strand formation, which reflects the relative stability of the base pair within the double-stranded structure. The larger the ΔG value, the more stable the double strand . Primers with a relatively high 5' end and intermediate ΔG values ​​and a low ΔG value at the 3' end (absolute value not exceeding 9) should be used. Primer 3 'end △ G value is too high, the structure is easy to form a double stranded site of the mismatch DNA and initiate a polymerization reaction. (The ΔG value at different locations can be analyzed with Oligo 6 software) 9. The 5' end of the primer can be modified while the 3' end is not modified. The 5' end of the primer determines the length of the PCR product, which has little effect on the specificity of the amplification. Therefore, it can be modified without affecting the specificity of amplification. The 5' end modification of the primer includes: adding a cleavage site; labeling biotin, fluorescence, digoxin, Eu3+, etc.; introducing a protein-binding DNA sequence; introducing a point mutation, an insertion mutation, a deletion mutation sequence; introducing a promoter sequence, and the like. The extension of the primer starts from the 3' end and cannot be modified. There is also no possibility of forming any secondary structure at the 3' end. 10. The single strand of the amplified product does not form a secondary structure. The main reason for the inefficiency of some primers is the influence of the single-stranded secondary structure of the amplified product. It is best to avoid the secondary structure region when selecting the amplified fragment. Relevant software (such as RNAstructure) can be used to predict the stable secondary structure of the mRNA and to help select the template. Experiments have shown that when the free energy ( ΔG °) of the region to be expanded is less than 58.6 l kJmol, the amplification is often unsuccessful. Substituting 7-deaza-2'-deoxy GTP for dGTP can be helpful in the success of amplification if this region cannot be avoided . 11. Primers should be specific. After the primer design is complete, it should be tested for BLAST . If you are not complementary to other genes, you can proceed to the next experiment. It is worth mentioning that the primer design of various templates is difficult. Some templates are difficult to condition themselves, such as high or low GC content, leading to find Primers that are not suitable for various indicators; PCR for cloning purposes, because the product sequence is relatively fixed, the selection of primers has a lower degree of freedom of choice. In this case, you can only retreat to the next level and try to meet the conditions. Yancheng Rongtai Labware Co.,Ltd , https://www.shtestlab.com