Endress+Hauser Analytik Jena Biometra TRIO - Page 75

Biometra TRIO Protocol Wizard
75
1. For each primer the melting temperature is calculated using the following equation
according to Wallace et al.
1
:
T
m
= ((w*A + x*T)*2) + ((y*G + z*C)*4)
where w, x, y and z are the number of the bases A, T, G and C in the primer se-
quence, respectively.
2. The average T
m
value is calculated from the T
m
values for both primer sequences
3. The annealing temperature T
a
is calculated based on the average T
m
value by apply-
ing the following rules:
 If the difference between the primer T
m
values is 4°C or less, the T
a
is calculated
as the average T
m
value minus 5°C
 If the difference between the primer T
m
values is higher than 4°C, the T
a
is calcu-
lated as the lower T
m
value plus 2°C.
For long primer sequences of 15 nucleotides or more the software calculates the primer
annealing temperature (T
a
) based on the nearest neighbor method according to Santa-
Lucia
2
and von Ahsen et al.
3
.
1
Wallace et al., Nucleic Acids Res. 6, 3543, 1979
2
SantaLucia, Proc Natl Acad Sci U S A. 1998 Feb 17; 95(4):1460-5
3
von Ahsen et al., Clin Chem. 1999 Dec; 45(12):2094-101