- Amplicon length: small amplicon = high amplification efficiency. Better would be less than 300 bp (ideal would be 100-200 bp)
- Amplicon Secondary Structure
- Avoid palindromes
- Avoid G/C rich areas
- Primer structure: avoid secondary structures (hairpins). Avoid repetitive sequences. Have less than four identical bases (primarily G’s)
- Avoid primers dimerisation (Forward/Forward, Reverse/Reverse or Forward/Reverse)
- Include 3 A’s or T’s in the last five 3’nucleotides
- Length 18 – 25 nt: Chance of finding a random primer binding site becomes more significant when primers are shorter.
- Tm value 50 – 60°C
- GC content ~ 45 – 55 %
- BLAST the primers for target specificity: http://www.ncbi.nlm.nih.gov/BLAST
- Desalted and HPLC purified primers are recommended. Purified primers provide increased specificity and therefore sensitivity; particularly important in SYBR® Green I reactions, as non-specific products contribute to SYBR® Green I fluorescence signal
- Primer3, funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute, is extremely reliable, fast, easy to use, and free.
- Use this link to download a PDF file of Probe/Primer design tips and guidelines
- Design exon-spanning primers
- Analyze primers for Tm, secondary structures and primer-dimer formation: GeneWalker