Advanced Services|Genotyping

Pimers (Re)Design

In some cases the switch from gel electrophoresis to HRM requires primers redesign.

To be on the safe side it is recommended to design separate reaction for WT and separate reaction for the target gene (mutant).

Here are the guidelines of Applied Biosystems for optimal Primers design:

  • Use amplicons with a target length of <300 (250) bp to ensure sensitive detection of sequence variants
  • Optimally, design three primer sets to empirically determine the most robust combination

Design attribute

Design guidelines

Amplicon

Length is less than 250 basepairs

Primer length

~20 bases each

Tm

58 °C to 60 °C (Optimal Tm is 59 °C)

% GC content

30–80% GC content in each primer

3’ end

No more than 2 G+C residues in the last 5 nucleotides at the3′ end

Repeating oligonucleotides

Avoid consecutive identical nucleotides. If you are unable to avoid consecutive identical nucleotides, make sure that each primer contains fewer than 4 consecutive Gs.