1. Tm Criteria: The primer melting temperature (Tm)
should be around 58-60 oC, and TaqMan® probe
Tm should be 10 oC higher than the Primer
Tm. The Tm of both the primers should
2. Length Criteria: Primers should be 15-30 bases in length.
3. GC Content: The
G+C content should ideally be 30-80%.
If a higher G+C content is unavoidable,
the use of high annealing and melting
temperature co-solvents such as glycerol
would be deemed essential.
4. GC Clamp: The total
number of Gs and Cs in the last five
nucleotides at the 3' end of the primer
should not exceed two. This helps to
introduce relative instability to the
3' end of primers to reduce non-specific
5. Amplicon Length: Maximum amplicon size should not exceed
400 bp (ideally 50-150 bases).
6. Runs and Repeats: The probes should not have runs of identical
nucleotides (especially four or more
consecutive Gs), G+C content should
be 30-80%, there should be more Cs than
Gs, and not a G at the 5' end.
7. Genomic DNA Avoidance: False-positive results are obtained
due to amplification of contaminating
genomic DNA. Thus,In the cDNA preparation,
it is preferable to have primers spanning