Description of the methods for DNA/DNA melting temperature calculations

For each oligonucleotide sequence, the melting temperatures were calculated using five different methods and/or parameters.

  • Basic Melting Temperature calculations are based in the following expression (Marmur and Doty, 1962): 

      where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

  ASSUMPTIONS:
  This equation assumes that the annealing occurs under standard conditions in a buffered solution of 50 mM Na+,
  50 nM of oligonucleotide concentration and with a pH near 7.0, though the Tm of DNA is unaffected within a
  significant range of pH around 7.0 due to the lack of titratable groups near this pH in Watson-Crick paired DNA.


  • Salt-adjusted Melting Temperature calculations are based in the following expression (Howley et al., 1979): 

       where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively. In this equation, the second 
   term adjusts for the GC content and the third term adjusts for the length of the sequence.

  ASSUMPTIONS:
  This equation also assumes that the annealing occurs under standard conditions of pH near 7.0 and 50 nM of 
  oligonucleotide concentration.


  • Nearest-neighbor thermodynamic Melting Temperature calculations are based in the following expression (Breslauer et al., 1986): 

       where:

         H (cal mole-1) is the sum of the nearest-neighbor enthalpy changes for hybrid
         formation (less than zero).

         A (cal K-1mole-1) is a constant for helix initiation which is equal to -10.8 cal
         K-1mole-1 for nonself-complementary sequences and = - 12.4 for self-complementary
         sequences).

         S (cal K-1mole-1)is the sum of the nearest-neighbor entropy changes for hybrid
         formation (less than zero)

         R is the molar gas constant (1.987 cal K-1mole-1)

         Ct is the total molar concentration of strands when oligonucleotides are not self
         complementary or it is equal to 4 times this concentration in the case of
         self-complementary sequences.

         In the nearest-neighbor formula, the two parameters affecting the Tm value are the salt
         concentration and the concentration of single-stranded DNA. These values are fixed at
         50 mM salt and 250 pM oligonucleotide.

  ASSUMPTIONS:
  The thermodynamic calculations assume that the annealing occurs in a buffered solution at pH near 7.0 and 
  that a two-state transition occurs.

    References

    Basic (bas) calculations were carried as described by Marmur, J. and Doty, P. (1962) J. Mol. Biol. 5, 109-118.

    Salt adjusted (salt) calculations were carried as described by Howley, P.M; Israel, M.F.; Law, M-F.; and M.A. Martin (1979) J. Biol. Chem. 254, 4876-4883.

    Nearest-neighbor thermodynamic 1 (Th1) calculations were carried out using the thermodynamic DNA parameters published by Breslauer, et al. (1986) PNAS USA 83, 3746-3750.

    Nearest-neighbor thermodynamic 2 (Th2) calculations were carried out using the thermodynamic DNA parameters published by Santalucia, et al. (1996) Biochemistry 35, 3555-3562.

    Nearest-neighbor thermodynamic 3 (Th3) calculations were carried out using the thermodynamic DNA parameters published by Sugimoto, et al. (1996) Nuc. Acid Res. vol 24, num 22, 4501-4505.