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duction
The
Tm, or melting temperature, characterizes
the stability of the DNA hybrid formed between
an oligonucleotide and its complementary
strand. The Tm is critical for determining
the optimal temperature at which to use
an oligonucleotide as a primer in PCR applications
(annealing temperature), as a probe for
in situ hybridization, and in Southern,
Northern or Dot blot analyses.finition
Tm
= Temperature at which 50% of a given oligonucleotide
is hybridized to its complementary strand.
In the absence of destabilizing agents,
like formamide or urea, Tm will depend on
3 major parameters:
-
The sequence: a GC-rich sequence has a
higher melting temperature.
-
The strand concentration: high oligonucleotide
concentrations favor hybrid formation,
which results in a higher melting temperature.
-
The salt concentration: high ionic strength
results in a higher Tm as cations stabilize
the DNA duplexes.trogen
Tm Calculation
The
most accurate method of estimating the Tm
of oligonucleotides, and that used at Retrogen,
is based upon a thermodynamic analysis of
the melting process from which it can be
shown that
| Tm
=( |
 |
S)+Rln(C))-273.15-12.0log[Na+]
|
|
| The
changes in enthalpy( |
|
H)
and entropy ( |
|
S)
of duplex formation are calculated from
|
nearest-neighbor thermodynamic parameters.
R is the molar gas constant (1.987 cal.K-1mole-1),
and C is the molar concentration of oligonucleotide.
Reference
-
Breslauer K.J., Frank R., Blocker H.,
Markey L.A. (1986) Predicting DNA duplex
stability from the base sequence - Proc.
Natl. Acad. Sci. USA 83, 3746-3750
-
Freier S.M., Kierzek R., Jaeger J.A.,
Sugimoto N., Caruthers M.H., Nielson T.,
Turner D.H. (1986) Improved free-energy
parameters for predictions of RNA duplex
stabilit. - Proc. Natl. Acad. Sci. USA
83, 9373-9377
-
Schildkraut C., Lifson S. (1965) Dependence
of the melting temperature of DNA on salt
concentration - Biopolymers 3, 195-208
-
Rychlik W. - Oligo version 4.0, Reference
Manual - National Biosciences, Inc., Plymouth,
MN
-
Rychlik W., Spencer W.J., Rhoads R.E.
- Nucleic Acids Res. 18, 6409-6412
-
Freier S.M. (1993) Hybridization: Considerations
affecting Antisense Drugs in Antisense
Research and Applications, eds. Grooke
S.T. and Lebleu B. CRC Press, Inc., 67-82
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