Primers:
Primers are artificially synthesized two-stage oligonucleotide sequences, one primer complementary to one DNA template strand at one end of the region of interest and the Other primer complementary to another DNA template strand at the other end of the region of interest. Importance of primers: Primers play an important role in the entire PCR system.
The importance of primers:
Primers play an important role in the entire PCR system. The specificity of PCR requires that the primer specifically binds to the target DNA and does not bind to other non-target DNA. The sensitivity of the PCR requires the DNA polymerase to effectively extend the primer. It can be seen that the primer design is closely related to the PCR result.
Primer design principles:
Primer length
Typically 15-30 nucleotides, longer primers should be used for long fragment PCR or for some specific PCR, but no more than 50 nucleotides.
2. Balance of base distribution
The same base should not appear more than 5 consecutively; the GC content is generally 40-60%; the GC content is too low, resulting in lower Tm value of the primer. The lower annealing temperature is not conducive to improving the specificity of PCR; the GC content is too high and easy. Initiates non-specific amplification.
3. Primer Tm value
General requirements: 55 ° C ~ 65 ° C.
Calculation:
For primers below 20 bases, the Tm value can be roughly estimated from Tm = 4 (GC) 2 (AT);
For longer primers, the Tm value needs to take into account the thermodynamic parameters, which are obtained from the “nearest neighbor†calculation, which is also the most commonly used calculation method for the existing primer design software.
Tm = â–³H/(â–³ SR * ln (C/4)) 16.6 log ([K ]/(1 0.7 [K ])) - 273.15
Primer secondary structure:
Primer dimers, as far as possible avoiding more base complementation at the 3' end between the two primer molecules.
Hairpin structure:
In particular, it is necessary to avoid the formation of a hairpin structure at the 3' end of the primer, which would otherwise seriously affect the extension of the DNA polymerase.
Primer 3' end:
The extension of the primer starts from the 3' end, so several bases at the 3' end need to be strictly paired with the template DNA, and no modification can be made, otherwise the effective extension cannot be performed, and even the PCR amplification completely fails. Given the degeneracy of the codon, the last base of the 3' end of the primer is preferably not paired with the third base of the codon.
Primer 5' end:
The 5' end of the primer may have a base that is not paired with the template DNA, and a non-template dependent sequence is introduced at the 5' end.
A restriction endonuclease site sequence was added to the 5' end (the 5' end of the restriction site plus the appropriate number of protected bases).
A site at the 5' end modifies a certain base and artificially introduces a point mutation at the site into the product for study.
The 5' end marks radioactive or non-radioactive substances (such as biotin, digoxin, etc.).
Internal stability of the primer:
In the past, it was thought that the 3' end of the primer should be firmly bonded to the template to effectively extend, so the 3' end is preferably G or C.
The current view is that the 5' end of the primer should be a relatively stable structure, and the 3' end is preferably a low-stability structure in the case of base pairing, that is, the 3' end should use A or T as much as possible, and use G or less. C.
A low-stability structure formed by only a few bases at the 3' end and a base at a non-specific site is difficult to effectively induce primer extension.
If the 3' end is a structure rich in G and C, only a few bases at the 3' end are complementary to the template, which may cause extension and cause false initiation.
Primer conservation and specificity
Conservative: universal primers - detect as many types of pathogenic microorganisms as possible;
Specificity: Avoid non-specific amplification.
According to its solubility, it can be divided into two types: water-soluble and water-insoluble.
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