Primer Selection
Specificity - %GC -
Dimer – Hairpin - Tm
Nucleotide
sequences
Amino Acid
sequences
CONSENSUS
Pileup
Pretty
Prettybox
Primer Selection Program-Prime
Amino Acid
Nucleotide
backtranslate
Confirm by BLAST
Targets an amplicon length of
75 to 150 bp
50 to 60% GC content
Limit secondary structure
Limit stretch of G or C’s
longer than 3 bases
No stable interaction between
forward and reverse
primers (primer/dimer
pairs)
Place C’s and G’s on ends of
primers,
but no more than 2 in the
last 5 bases on 3’ end
Melting Temperature (Tm) above
50 oC
Verify specificity