There should be no complementary sequences between primers themselves. There should be less than 4 consecutive complementary bases. The g value of primer should not be too high. The greater the g value, the more stable the double strand.

Primer length and GC content should be moderate. The length of the primer is about 18~28 bp, but it should not be greater than 38 bp. If the primer is too short, it will affect the specificity of amplification. If it is too long, its extension temperature will be greater than 74℃, which is not conducive to the catalytic reaction of Taq enzyme. If the amplified product is 4~5 kb, the primer should be no less than 24bp.

Extended data:

Precautions:

1, and the primer length is generally 15-30? Bp, commonly used as 18-27? (22)bp, but not more than 38, because too long will lead to its extension temperature greater than 74℃, not suitable for Taq? DNA polymerase reaction. ?

2. The bases should be randomly distributed: there should be no primer sequences with high similarity in the template, especially those with high similarity at the 3' end, otherwise it will easily lead to false? One way to reduce the similarity between primer and template is that the distribution of four bases in primer should be random, and there should be no purine and pyrimidine.

The 3,3' end should not exceed 3 consecutive G or C, such as GGG or CCC, because this will cause the primer to be wrongly triggered in the GC-rich sequence region.

4. The last base at the 3' end of the primer has a great influence on the DNA synthesis efficiency of Taq enzyme. The difference of the last base leads to different amplification efficiency of the mismatch position, and the mismatch efficiency of the last base A is obviously higher than the other three bases, so the base A at the 3' end of the primer should be avoided.

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