HeIIo dear aII:
Im aIready famiIiriazed with parameters needed for qpcr primer design, so I have made the design manuaIIy with PRIMER 3 and OIigoAnaIizer and with private software Primer express finding the foIIowing:
I chosed primers in Primer 3 which had a maximun dimer of deIta G= -6 and -1.6 in 3´ end. PRIMER EXPRESS have chosen primers wich have maximum deIta G= -8 and equaIIy Iess than -1.6 in 3´ end. The main difference is the deIivery of secondary estructures , Primer Express seems to deIiver better tempIate secondary estructures with Iess estabiIity than the manuaI design. Im not sure I shouId choose this primers since they have a dimer of -8 but its tempIate aIIows them to anneaI easiIIy.
I wouId reaIIy appreciate if someone can advice , and if someone can share their experience.
These are the factors I generally use when designing primers for RT-qPCR (I use PrimerBlast, by the way):
- Final amplicon size of 100-300 bp
- Approx. Tm of 60C
- Exon-exon junction spanning
- Hybridization towards the 3' of the gene
- Fewer than 3 C or G bases at the 3' end of each primer
This are some points that I keep in mind:
• Length: 18-30 bases
• GC content: 40-60 %
• Tm: 55-60 °C, ΔTm difference between forward primer and reverse primer should be ≤ 4 °C
• Avoid 3′ end T (allows mismatching)
• Avoid complementarities within the primers to avoid hairpins
• Avoid complementarities between the primers to avoid primer dimers, especially of 2 or more bases at the 3′ ends of the primers
• Check if primers are unique and specific (check with a BLAST search)
• Design intron spanning or intron flanking primers to prevent amplification of contaminating genomic DNA.
In your case, I would suggest you to design new primers.
I used the Beacon Designer software (trial 30 days) has many tools for designing primers and probes. When the software gives me the primers with best score I perform analysis of secondary structure formation and the temperatures of these structures near the annealing ARE NOT primers and delta G are positive values. Also I realize Blast analysis.
You could corroborate your design with the Beacon Designer or Gene Runner latter is free
You don't want the primer binding to any secondary structure in your template and the required annealing temperature. Use DNAfold http://mfold.rna.albany.edu/?q=mfold/dna-folding-form to check for secondary structure in your template. Choose another primer if it binds within a hairpin structure on your template at the annealing temperature.
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