Thermodynamics of DNA hybridization

 

A combination of spectroscopy and calorimetry was used to determine the free energies of melting of short oligonucleotides.  Based on these measurements the free  energy of a helix can be determined based on10 sets of nearest-neighbor pairs shown in Figure 16.5.  In addition to these values we need to know the free energy of the initiation (i.e. the first base pair).  The overall free energy is then calculated from:

 

DGo = DGo (initiation) + S DGo (nearest neighbors)

 

Sample problem. Determine the melt temperature for the oligonucleotide

 

                     5’-ATAGCA-3’

                                                     ß              5’-ATAGCA-3’

                                                         à          3’-TATCGT-5’

                      3’-TATCGT-5’

 Solution:

    DGo = DGo (initiation) + S DGo (nearest neighbors)

=    20.9          - 6.3            - 3.8            - 6.7           - 13.0            - 6.9

=   -16.8 kJ

Note that we use GC initiation if there is a single GC base pair.

Only use AT initiation of the strands are all A and T.

 

    DGo = -16.8 kJ

Notice that the the free energy of initiation is positive.  Initiation is unfavorable because of the entropy that must be overcome to bring the chains together.  To calculate the melt temperature we need the enthalpy of reaction as well.

        =  - 36.0           - 25.1            - 32.6           - 46.4            - 24.3

        = -164.4 kJ

 

 

Slide1.BMPSlide2.BMP

Figure 1. Thermodynamic data for base pair formation.  Each possible combination of base pairs is given both for initiation and for internal (nearest neighbor) configurations.  Data were obtained from Breslauer et al. PNAS, 93, 3746 (1986).

 

The data in Figure 1 can be used to estimate the stability of a mutagenesis primer.  However, it can also be applied to the determination of the melt temperature of a primer.  The quantities DGo, DHo and DSo are tabulated at 298 K.  The hybridization free energy, DGo, decreases as the temperature is increased according to,

 

 

At the melt temperature, Tm,    Therefore,

 

 

which implies that

 

Thus, the values in the thermodynamic tables can also be used to estimate the melt temperature of an oligonucleotide.  Such determinations are now routine in computer programs for commercial primer design for mutagenesis protocols using the polymerase chain reaction.