Techne Prime Elite User Manual
Page 24
22
OLIGONUCLEOTIDE T
M
CALCULATOR
One of the most important parameters in a PCR is the annealing temperature of the reaction. This can
be determined theoretically using various software programmes to calculate the melting temperature
(T
m
) of the primers.
The T
m
is defined as the temperature at which half the DNA strands are single stranded and half are
double stranded when base paired to a complimentary strand. Using annealing temperatures that are
well below the T
m
of the primers can result in mismatching, false priming and may lead to primer-dimer
artefacts. Annealing temperatures that are significantly higher than the primer T
m
may result in
reduced priming or prevent priming altogether.
The Prime Elite uses the Nearest-Neighbour method for calculating the T
m
of an oligonucleotide
1
.
The T
m
is calculated using the following equation:
T
m
=
{(∆Hº x 1000)/(A + ∆Sº + R ln(C/4))} - 273.15 + 16.6 x log[Na
+
]
Where:
∆Hº =
The sum of the Nearest-Neighbour enthalpy changes (Kcal/mol)
A
=
Helix initiation correction constant
∆Sº =
The sum of the Nearest-Neighbour entropy changes (cal K
-1
mol
-1
)
R
=
The Gas Constant (1.99 cal K
-1
mol
-1
)
C
=
Concentration of the oligo (M)
[Na+] =
Concentration of monovalent ions (M)
1
Breslauer, K.J.; Frank, R.; Blocker, H. and Marky, L.A. (1986) Proc. Natl. Acad. Sci. USA 83, pp
3746-3750.
The Oligonucleotide T
m
calculator can be found in the Settings module where the user can input the
oligonucleotide sequence, its concentration in the reaction and the salt concentration of the reaction
mix.