Appendix Ii: Factors Affecting Stringency Of Hybridisation Reactions - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

FACTORS AFFECTING STRINGENCY OF
The stringency of Hybridisation determines the degree to which mismatched hybrids are
permitted to form. The optimum Hybridisation temperature for DNA - DNA Hybridisations
is 25°C below the melting temperature (Tm).
The Tm is affected by: -
1. The concentration of monovalent cation (M).
2. Base composition (percentage G+C content).
3. The length of the duplex in base pairs (L).
4. The amount of formamide in the reaction mixture (F).
These variables have been linked by the following equation (see Meinkoth and Wahl,
1984): -
Tm = 81.5°C + 16.6 logM + 0.41 (% G+C) - 0.61 (% F) - 500/L
This equation relates to probes longer than approximately 50 nucleotides (see Chapter 6
for conditions relating to oligonucleotide Hybridisations).
As an illustration, in a reaction carried out in a solution of 6 x SSC and no formamide,
with a 50% GC rich 500bp probe, the Tm is calculated to be 101°C (i.e. Hybridisation
temperature 75°C).
Substituting the 6 x SSC with 1 x SSX in this system (i.e. during washing), reduces the
Tm by 13°C. Including 50% formamide in each solution would have the effect of
reducing each Tm by 30.5°C. A shorter probe length (50bp) reduces the Tm by a further
10°C.
In practice, it is not necessary to calculate the Tm accurately for every system, and the
standard conditions given in the protocol sections will give good results. However, the
equation does give a useful indication of the effect of adjusting the different variables on
the stringency. In general, hybridise at low stringency; then during washing, increase the
stringency by keeping the temperature constant but decreasing the salt concentration
with each wash step.
© Thermo Scientific, May 2003. Issue 7
APPENDIX II
HYBRIDISATION REACTIONS
32