HYBRIDIZATION NOTES - Melting and Annealing Temperature

A va riety of factors affect the efficiency of hybridization between two strands of DNA. These include the nature of the hybridizing molecules (DNA or RNA), their lengths, the hybridization environment (salt concentrations and denaturants), probe concentrations, and their sequences.

For membrane bound targets and moderately long DNA probes, Howley et al1 determined that the melting temperature (Tm) at which 50% of a probe is annealed to its complementary strand is defined by:

Tm = 81.5 + 16.6logM + 41(%G + %C) - 500/L - 0.62F
where
M = molar concentration of monovalent cations
%XG or C = the respective fraction of G and C nucleotides in the probe
L = length of the annealed product
F = molar concentration of formamide
For example, a short oligonucleotide probe with the sequence AGGTCATTG in a 75 mM solution without formamide has a predicted Tm = 81.5 + 16.6log(0.075) + 41(0.33+0.11) - 500/9 - 0.62(0)
                     = 24.1°C
This equation is inappropriate for probes less than about 50 nucleotides. Modifications of this equation include,
        Tm of RNA = 79.8+18.5logM+58.4(%G+%C)+11.8(%G+%C)2-820/L-0.35F
        Tm of an RNA-DNA hybrid = 79.8+18.5logM+58.4(%G+%C)+11.8(%G+%C)2-820/L-0.50F
The larger numbers reflect the increased stability of hybrids formed with RNA.

For oligonucleotides, Wallace, et al2 determined that
Td=2(A+T)+4(C+G), where Td = temperature (in °C) at which 50% of the oligonucleotides are annealed to their membrane-bound complementary sequences. The number of each particular nucleot ide in the probe is inserted into the equation in place of the letters. The equation is useful for short (14-20 mers) in 0.9 M NaCl.

        ex: for sequence AGGTCATTG, the Td = 2(2+3)+4(1+3) = 26°C
When the target and probe are free in solution, add 7-8°C to Td.

Melting temperature in solution is determined by plotting O. D versus temperature. The mid point on the S-shaped curve is the melting temperature.

Other estimates of melting points have been determined for DNA3 or RNA4 based on nearest neighbor analysis (reviewed by Genosys5). Breslauer, et al3 showed that melting behavior of a DNA duplex is predictable from its primary sequence.
Here,

Tm = 1000(DH)/[A+DS)+Rln(Ct/4)]-273.15+16.6log(Na+)].
where
DH = the sum of nearest neighbor enthalpy changes moving one base a t a time through the sequence
A = correction for initiation of pairing (= -10.8)
DS = the sum of nearest neighbor entropy changes
R = 1.987 cal deg-1 mol-1)
Ct is the molar concentration of strands.
For self-complementary strands, the term "Ct/4" is replaced by Ct.
The values for DH and DS are shown in the table.
Nearest Neighbor DH DNA (kcal/mol) DH RNA (kcal/mol) DS DNA (cal/mol); DNA DS RNA (cal/mol)
AA or TT - 9.1 - 6.6 -24.0 -18.4
AT - 8.6 - 5.7 -23.9 -15.5
TA - 6.0 - 8.1 -16.9 -22.6
CA or TG - 5.8 -10.5 -12.9 -27.8
GT or AC - 6.5 -10.2 -17.3 -26.2
CT or AG - 7.8 - 7.6 -20.8 -19.2
GA or TC - 5.6 -13.3 -13.5 -35.5
CG -11.9 - 8.0 -27.8 -19.4
GC -11.1 -14.2 -26.7 -34.9
GG -11.0 -12.2 -26.6 -29.7

As an example, a 1 µM solution of the probe mentioned above (AGGTCATTG) in a 150 mM solution has a predicted Tm of:
        Tm = 1000(-7.8-11.0-6.5-5.6-5.8-8.6-9.1-5.8) / [-10.8+(-20.8-26.6-17.3—13.5-12.9-23.9-24.0-12.9) + 1.987ln[(1E-06)/4]] - 273.15 + 16.6log(0.15)
              = [-60900 / (-10.8-151.9-30.2)] - 273.15 - 13.7
              = (-60900/-192.9) - 286.9
       &n bsp;      = 29°C

The applicability of these equations to laboratory situations varies as additional components in the hybridization environment are altered. It is best to consider these predictions guidelines, since they may vary for particular sequences from empirically derived determinations.


Background Problems

Moderate background on filter hybridizations is common. They often can be reduced by washing in up to 7% SDS.


References
1. Howley, P.M., Israel, M.F., Law, M.F., and Martin, M.A. J. Biol. Chem. 254:4876.
2. Wallace,R.B., Shaffer,J., Murphy,R.F., Banner,J., Hirose,T., and Itakura,K. Nucl. Acids Res. 6:3543, (1979).
3. Breslauer, K.J., Frank, R., Blocker, H., and Marky, L.A. PNAS 83:3746-3750 (1986).
4. Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T. and Turner, D.H. PNAS 83:9373-9377 (1986).
5. Transcripts : Melting Temperature. Genosys Biotechnologies, Inc., Woodlands, TX.
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Proper citation for data acquired from this document is: "Frank, M. B. Hybridization Notes - Melting and Annealing Temperature. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/hybnotes.html). 1997. Oklahoma City. Revision Date: October 2, 1997."