Primer design factors to consider, heterozygous such as: primer length (primer heterozygous lengt

Vietnamese beauty stunning large-scale behavior (Figure primer design principles to keep the template primer closely; you can not have a stable hairpin dimers or between primer and primer; heterozygous primer not cause efficient in other non-target sites DNA polymerization (ie mismatch).
Primer design factors to consider, heterozygous such as: primer length (primer heterozygous length), product length heterozygous (product length), sequence Tm value (melting temperature), ΔG values (internal stability), primer-dimer and hairpin (duplex formation and hairpin), error priming site (false priming site), primer and product GC content (composition), and sometimes also on the modified primers, such as increasing heterozygous the restriction point, the introduction of mutations.
Primer design elements General primer length 16-23bp, commonly used length heterozygous 18-21bp, too long or too short are inappropriate. primer 3 'end heterozygous of the base generally do A, because A is caused by an error caused by the relatively high efficiency of the sites, while the other three bases of error caused by a relatively small number of efficiency. primers GC content is generally heterozygous 45-55% too high or too low is not conducive to initiate the reaction. GC content of the upstream and downstream primers can not be that much difference. Tm value template sequence corresponding primers preferably about 72 , of course, due to the composition of the template sequence itself determine heterozygous its Tm value may be low or high, depending on the circumstances can be applied flexibly. ΔG value reflects the degree of intensity of the primer and template binding, is also an important primer evaluation. In general, the Oligo 5.0 software ΔG value window, ΔG value of the best primers sinusoidal shape, namely the 5 'end and the middle part of ΔG value is higher, while the 3' end of the ΔG values are relatively low, and do not超过 9 (ΔG is negative, there is absolute value), so it will help to prevent and correct errors caused by initiating the reaction. principle, primer template should have a high bond energy, it is a good integration of the primer to the template sequence, thus the 5 'end and ΔG values should be higher heterozygous intermediate section, and the 3' end of the DNA polymerase of the impact value ΔG melting the template DNA, this step is not conducive to high possible errors priming site depends on the composition of the primer sequence and the template sequences of similarity, the similarity is high, the error rate caused, the error rate caused by initiation generally do higher than 100, preferably no false priming sites, so a non-desired product can guarantee no false band. Energy primer dimer and hairpin structures heterozygous generally do not exceed 4.5, or prone to primer dimer band, and that will reduce the concentration resulting PCR primers can not be a normal heterozygous reaction. modification of the primer is generally increased restriction sites should refer to the carrier restriction enzyme recognition sequence determination of the downstream primer often modified sequence chosen a different restriction enzyme recognition sequence to facilitate future work. Automatic search and evaluation on the primer analysis We recommend using automatic search software: Primer Premier 5.0 Recommended evaluation heterozygous software using primers: Oligo 5/6
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