primers complementary to itself can not have four consecutive esco bases.

primers complementary to itself can not have four consecutive esco bases.
PCR primer design purpose is to find a pair of appropriate nucleotide fragments, so that it can effectively amplify template DNA sequences. Such as the merits of the foregoing, the primer is directly related to the specificity of PCR and success. For primer esco design esco can not have an all-encompassing rule to ensure the success of PCR, but follow certain principles, it helps to design primers. 1. The primers specific primers and non-specific amplification sequence homology of 70% or not more than eight consecutive complementary esco bases homologous. Some primer 2. The main reason for avoiding ineffective products that affect secondary structure region primer DNA secondary structure of the repeat region, the amplified fragment is best to avoid the selection of regions of secondary structure. With related esco computer software esco can predict the stability of mRNA secondary structure is estimated to help select a template. Experiments show that the area be extended free energy esco ( G ) less than 58.6lkJ / mol, the amplification is often not successful. If not avoid this area, with a 7-deaza-2'- deoxy-GTP replace esco dGTP amplification success is helpful. 3. The length of the oligonucleotide primers of length 15 ~ 30bp, generally 20 ~ 27mer. The effective length of the primer: Ln = 2 (G + C) + (A + T +, Ln value can not be greater than 38, as> 38, the optimum stretching temperature exceeds the optimum esco temperature of Taq DNA polymerase (74 ), can not ensure product specificity. esco 4.G + C content of G + C content is generally 40% to 60% of its value Tm is the melting temperature of the oligonucleotide, i.e., salt concentration, under certain conditions, 50% of oligonucleotides temperature acid double strand, the effective start-up temperature, generally higher than the Tm value of 5 ~ 10 . If according to the formula esco Tm = 4 (G + C) +2 (A + T) estimated Tm value of the primer, the effective primer Tm of 55 ~ 80 , which is preferably close to 72 Tm value so that optimal refolding conditions. 5. randomly distributed alkali base distribution of the four bases in the primer is preferably random, do not have a poly-purine esco or pyrimidine poly The presence of particular 3 'end should not be more than three consecutive G or C, because this would allow the primers in G + C rich sequence regions false priming. 6. The primer itself primer complementary sequence itself should not exist, otherwise, the primer itself may folded into a hairpin structure itself dental primer annealing. This secondary structure due to steric affect primer template complex combination. If using human judgment, primers complementary bases in a row can not be greater than itself 3bp. 7. Primer should not complementary nature between the two primers, especially should avoid the 3 'end of the complementary overlap between primer dimer formation esco in case. A pair of primers should be more than four consecutive nucleotides homologous or complementary. esco 'end of the primer extension from the 3' end of the primer 3 8. started, can not make any modifications .3 'end can not have any secondary structure may be formed, in addition to the specific PCR (AS-PCR) reaction, the primer 3 'end of the mismatch can not occur in a standard PCR reaction, with 2U Taq DNA polymerase and 800μmol / L dNTP (four kinds of dNTP each 200μmol / L) plasmid (103 copies) as a template, 95 , 25s.; 55 , 25s; 72 , 1min amplification cycle parameters under HIV-1 gag gene region, the primer 3 'end of mismatching Amplified Products is a certain mismatch .A:A regular production declined to make fall 1/20, A:G and C:C wrong to 1/100 seven primers A:. template primer G G: A mismatch on PCR template effects are equivalent 9. primer 5 'primer 5'-end. esco defining a length of the PCR product were not amplified specifically affect Thus it can be modified without affecting the specificity of amplification primers 5 'end modifications include: esco add restriction sites; labeled biotin, fluorescence, digoxin, Eu3 +, etc; introduction esco of protein-bound DNA sequences; introducing mutations, insertions and deletions sequences and promoter sequences such as the introduction of a 10 codon degeneracy as amplified coding regions, primer 3 'end not terminated. Bit 3 in codon, because the first three easily degenerate codon occurs and will affect amplification specificity esco and efficiency. esco primer design special purpose will be discussed in the relevant sections. As people know primer, some primers computer design programs have emerged, the following will discuss computer primer design methods.