These three software can handle PCR primers design
Today we're going to summarize the software or websites that design the primers, and the common ones.
In the PCR process, primer design is a very important link, good primers will make your PCR experiment twice the result!
PCR primers design software
Primer design 1: primer Premier
Main applications: the design of PCR or sequencing primer and hybrid probe, software for evaluation;
Four main function points, primer design, analysis, DNA restriction enzymes loci primitives (motif) search and homology analysis function, design degenerate primers, "reading", DNA and protein sequences of swaps, voice prompt keyboard input, etc.
Primer design 2:Oligo software
Main applications: the design of nucleic acid sequence primer analysis and the calculation of hybrid temperature (Tm) and theoretical prediction sequence of nucleic acid sequence.
Main functions: search of common primer, design of sequencing primer, design of hybrid probe, and evaluation of primer on quality, etc.
Primer design 3:PrimerExpress
Main applications: NT oligonucleotide design procedure, the software can design standard DNAPCR primers, rt-pcr primers, nested PCR primers, allele specific primers, multiple PCR primers with TaqMan probe using DNAPCR primers.
The software has a primer inspection system, a primer annotation, and a TaqMan MGB probe and TaqMan probe can be designed for a single sequence and multi-sequence probe.
Note the key points of the primer design
Basic principles
(1) the sequence of primers and templates should be closely complementary
(2) to avoid forming a stable dimer or hairpin structure between primers and primers
(3) the primer cannot trigger the DNA polymerization reaction at the non-target site of the template
Matters needing attention
1.Primers length, generally between 15 ~ 30 bases, commonly used is 18 to 27 bp, but should not be more than 38, because is too long can lead to the extension temperature greater than 74 ℃, not suitable for Taq DNA polymerase reaction.
2.The sequence of primers should not be similar in the template, especially the sequence with high similarity of 3 'end, which can easily lead to mismatches.
3.The GC content of the primer sequence is generally 40-60%, too high or too low is not conducive to the reaction.
In addition, the GC content of upstream and downstream primer should not be too different.
4.There is no Annealing between the two primers for the primer design. It is necessary to pay special attention to the 3 'terminates. The 5' end sequence does not have too much influence on the PCR, so it is commonly used to introduce modification sites or markers.
5.Primers corresponding template sequence Tm value at about 72 ℃ can make the best operating conditions.
Tm values are calculated in A number of ways, such as by formula Tm = 4 (G + C) + 2 (A + T), which is used in the Oligo software (the nearest neighbor method).