I |
length fragment |
< 100 bp |
<300bp |
|
length primers |
17-25 bp |
17-30 bp |
II |
Theoretical melt temperature |
>55°C -
<60°C |
>50°C - <65°C |
|
range of variation in melt temperature for developed toolkit |
< 5 °C |
5 °C - 10 °C |
III |
GC% |
40-60 |
30-70 |
IV |
ΔG hairpin, self-dimer, hetero dimer |
< 6.00 |
< 9.00 |
|
bases suitable for binding with own primer sequence |
<6 |
<10 |
V |
BLAST identity |
>90% |
>99% |
VI |
Mismatches both primer site closely related species
(Ireland) |
>10 |
>6 |
|
Mismatches each primer site (within first 5 basepairs on 3’) |
>3 |
>1 |