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The purpose of PCR primer design is to find a pair of suitable nucleotide fragments that can efficiently amplify the template DNA sequence
.
As mentioned above, the quality of primers is directly related to the specificity and success of PCR
.
There can be no one-size-fits-all rule for primer design to ensure the success of PCR, but following certain principles can help with primer design
.
1.
The specificity of primers The homology of primers and non-specific amplification sequences should not exceed 70% or homology of 8 consecutive complementary bases
.
2.
Avoid the secondary structure region of the product The main reason for the ineffectiveness of some primers is the influence of the secondary structure of the DNA in the repeat region of the primer.
It is better to avoid the secondary structure region when selecting amplified fragments
.
The stable secondary structure of mRNA can be predicted and estimated by relevant computer software, which is helpful for selecting templates
.
Experiments show that when the free energy of the region to be expanded (△G°) is less than 58.
6 lkJ/mol, the amplification is often unsuccessful
.
If this region cannot be avoided, replacement of dGTP with 7-deaza-2'-deoxyGTP can be helpful for successful amplification
.
3.
Length The length of oligonucleotide primer is 15~30 bp, generally 20~27mer
.
Effective length of primer: Ln=2(G+C)+(A+T+, Ln value cannot be greater than 38, because when >38, the optimal extension temperature will exceed the optimal temperature of TaqDNA polymerase (74℃), it cannot be guaranteed 4.
G+C content G+C content is generally 40%~60 %
.
The Tm value is the melting temperature of the oligonucleotide, that is, under a certain salt concentration, the temperature at which 50% of the double strands of the oligonucleotide melts, and the effective starting temperature is generally 5~10°C higher than the Tm value
.
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℃, and its Tm value should preferably be close to 72℃ to make the best annealing condition
.
5.
Random distribution of bases The distribution of the four bases in the primers should be random, without the presence of polypurines or polypyrimidines
.
In particular, there should be no more than 3 consecutive Gs or Cs at the 3' end, as this will cause the primer to misprime in the G+C-rich sequence region
.
6.
The primer itself The primer itself should not have complementary sequences, otherwise the primer itself will fold into a hairpin-like structure and the primer itself will renature
.
This secondary structure will affect the renaturation of primers and templates due to steric hindrance
.
If artificial judgment is used, the continuous complementary base of the primer itself cannot be larger than 3bp
.
7.
Between primers The two primers should not be uncomplementary, especially the complementary overlap at the 3' end should be avoided to prevent the formation of primer dimers
.
There should be no more than 4 consecutive bases of homology or complementarity between a pair of primers
.
8.
The 3' end of the primer The extension of the primer starts from the 3' end, and no modification can be made
.
There is also no possibility of forming any secondary structure at the 3' end, except in a special PCR (AS-PCR) reaction, the 3' end of the primer cannot be mismatched
.
In a standard PCR reaction system, 2UTaq DNA polymerase and 800 μmol/L dNTPs (200 μmol/L for each of the four dNTPs) were used as templates with plasmid (103 copies) as a template, and the cycles were 95°C, 25s; 55°C, 25s; 72°C, 1 min.
Under the condition of parametric amplification of HIV-1gag gene region, the effect of primer 3' end mismatch on the amplified product is regular
.
A:A mismatch reduced the yield to 1/20, and A:G and C:C mismatches decreased to 1/100
.
Primer A: template G and primer G: template A mismatches have the same effect on PCR
.
9.
The 5' end of the primer The 5' end of the primer defines the length of the PCR product, which has little effect on the specificity of amplification
.
Therefore, it can be modified without affecting the specificity of the amplification
.
The modification of the 5' end of the primer includes: adding an enzyme cleavage site; labeling biotin, fluorescence, Eu3+, etc.
; introducing a protein-binding DNA sequence; introducing a mutation site, insertion and deletion mutation sequences, and introduction of a promoter sequence
.
10.
Codon degeneracy If the coding region is amplified, the 3' end of the primer should not be terminated at the 3rd position of the codon, because the 3rd position of the codon is prone to degeneracy, which will affect the specificity and efficiency of amplification
.
Special purpose primer design is discussed in the relevant section
.
With the understanding of primers, some computer design programs for primers have emerged.
The following will discuss the computer design methods of primers
.
.
As mentioned above, the quality of primers is directly related to the specificity and success of PCR
.
There can be no one-size-fits-all rule for primer design to ensure the success of PCR, but following certain principles can help with primer design
.
1.
The specificity of primers The homology of primers and non-specific amplification sequences should not exceed 70% or homology of 8 consecutive complementary bases
.
2.
Avoid the secondary structure region of the product The main reason for the ineffectiveness of some primers is the influence of the secondary structure of the DNA in the repeat region of the primer.
It is better to avoid the secondary structure region when selecting amplified fragments
.
The stable secondary structure of mRNA can be predicted and estimated by relevant computer software, which is helpful for selecting templates
.
Experiments show that when the free energy of the region to be expanded (△G°) is less than 58.
6 lkJ/mol, the amplification is often unsuccessful
.
If this region cannot be avoided, replacement of dGTP with 7-deaza-2'-deoxyGTP can be helpful for successful amplification
.
3.
Length The length of oligonucleotide primer is 15~30 bp, generally 20~27mer
.
Effective length of primer: Ln=2(G+C)+(A+T+, Ln value cannot be greater than 38, because when >38, the optimal extension temperature will exceed the optimal temperature of TaqDNA polymerase (74℃), it cannot be guaranteed 4.
G+C content G+C content is generally 40%~60 %
.
The Tm value is the melting temperature of the oligonucleotide, that is, under a certain salt concentration, the temperature at which 50% of the double strands of the oligonucleotide melts, and the effective starting temperature is generally 5~10°C higher than the Tm value
.
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℃, and its Tm value should preferably be close to 72℃ to make the best annealing condition
.
5.
Random distribution of bases The distribution of the four bases in the primers should be random, without the presence of polypurines or polypyrimidines
.
In particular, there should be no more than 3 consecutive Gs or Cs at the 3' end, as this will cause the primer to misprime in the G+C-rich sequence region
.
6.
The primer itself The primer itself should not have complementary sequences, otherwise the primer itself will fold into a hairpin-like structure and the primer itself will renature
.
This secondary structure will affect the renaturation of primers and templates due to steric hindrance
.
If artificial judgment is used, the continuous complementary base of the primer itself cannot be larger than 3bp
.
7.
Between primers The two primers should not be uncomplementary, especially the complementary overlap at the 3' end should be avoided to prevent the formation of primer dimers
.
There should be no more than 4 consecutive bases of homology or complementarity between a pair of primers
.
8.
The 3' end of the primer The extension of the primer starts from the 3' end, and no modification can be made
.
There is also no possibility of forming any secondary structure at the 3' end, except in a special PCR (AS-PCR) reaction, the 3' end of the primer cannot be mismatched
.
In a standard PCR reaction system, 2UTaq DNA polymerase and 800 μmol/L dNTPs (200 μmol/L for each of the four dNTPs) were used as templates with plasmid (103 copies) as a template, and the cycles were 95°C, 25s; 55°C, 25s; 72°C, 1 min.
Under the condition of parametric amplification of HIV-1gag gene region, the effect of primer 3' end mismatch on the amplified product is regular
.
A:A mismatch reduced the yield to 1/20, and A:G and C:C mismatches decreased to 1/100
.
Primer A: template G and primer G: template A mismatches have the same effect on PCR
.
9.
The 5' end of the primer The 5' end of the primer defines the length of the PCR product, which has little effect on the specificity of amplification
.
Therefore, it can be modified without affecting the specificity of the amplification
.
The modification of the 5' end of the primer includes: adding an enzyme cleavage site; labeling biotin, fluorescence, Eu3+, etc.
; introducing a protein-binding DNA sequence; introducing a mutation site, insertion and deletion mutation sequences, and introduction of a promoter sequence
.
10.
Codon degeneracy If the coding region is amplified, the 3' end of the primer should not be terminated at the 3rd position of the codon, because the 3rd position of the codon is prone to degeneracy, which will affect the specificity and efficiency of amplification
.
Special purpose primer design is discussed in the relevant section
.
With the understanding of primers, some computer design programs for primers have emerged.
The following will discuss the computer design methods of primers
.
