Molecular Biology Enzymes

Equations for calculating Tm of nucleic acids

1. DNA-DNA hybrids (holds for Na+ concentrations between 0,01 and 0,4 M and %GC values of 30-75%),(a) Maniatis et al., 1982; (b) Bolton, E.T., McCarthy, B.J. (1962) Proc. Natl. Acad. Sci. USA 48, 1390-1397.

(a) Tm = 81,5°C + 16,6 · log10[Na+] + 0,41 · %GC - 600/n,

(b) Tm = 81,5°C + 16,6 · log[Na+] + 0,41 · %GC - 500/n - 0,65 · %F,

where Tm is the melting temperature; [Na+] is the molar sodium concentration; %GC is the percent of Gs and Cs in the sequence ; n is the length of the sequence; %F is the percentage of formamide in the hybridization solution.

2. DNA-RNA hybrids (holds for Na+ concentrations between 0,01 and 0,4 M and %GC values of 30-75%), Casey, J., Davidson, N. (1977) Nucleic Acids Res. 4, 1539.

Tm = 79,8°C + 18,5 · logM + 0,58 · %GC + 11,8 · %GC² - 0,5 · %F - 820/n,

where Tm is the melting temperature; M is the molarity of monovalent cations (Na+ concentration); %GC is the percent of Gs and Cs in the sequence ; %F is the percentage of formamide in the hybridization solution; n is the length of the duplex sequence.

3. RNA-RNA hybrids (holds for Na+ concentrations between 0,01 and 0,4 M and %GC values of 30-75%), Bodkin, D.K., Knudson, D.L. (1985) J. Virol. Methods 10, 45.

Tm = 79,8°C + 18,5 · logM + 0,58 · %GC + 11,8 · %GC² - 0,35 · %F - 820/n,

4. DNA oligonucleotides (for duplex oligonucleotides 14- 25 bp in length), Wallace, R.B. et al. (1979) Nucleic acids Res. 6, 3543.

Tm = 2 · (ndA + ndT) + 4 · (ndC + ndG),

where Tm is the melting temperature; n is a number of a corresponding base in oligonucleotide. Presence of m⁵C in oligonucleotide increases the Tm of duplex; m⁴C and m⁶A have an opposite effect (Butkus, V. et al. (1987) Nucleic Acids Res. 20, 8467-8478; Jurgaitis, A. et al. (1988) Bioorganicheskaya Khimiya 14, 158-165).

5. Factors influencing the Tm of nucleic acid hybrids

Each 1% formamide reduces the Tm by about 0,6°C. 6M urea reduces the Tm by about 30°C. The Tm is reduced by 1°C for each 1% of mismatching. Tm increases 16,6°C for each onefold increase in monovalent cations, between 0,01 and 0,40 NaCl. Compatible solutes, such as ectoin and betain, at a final concentration of 1-1,7 M reduces melting temperature of GC-rich DNA up to 6°C and improves the amplification of GC-rich DNA or templates that form strong secondary structures (e.g. repeats). DMSO effective lowers the melting and strand separation temperatures (by 5°-6°C per 10% DMSO).