There is a linear relationship between the base composition of DNA as expressed in terms of the guanine plus cytosine bases and the denaturation (Tm, or thermal melting) temperature. E coli DNA serves as a reference material of known composition.
mole percent guanine plus cytosine =
= ((Tm of sample in °C) + ((90.5°C) - (Tm of reference E. coli DNA sample in °C)) - 69.3) / 0.41
where:
• 90.5°C is the melting point of E. coli DNA in 1x SSC
• E coli DNA has a G+C content of 51 mol%
Technical points:
• The melting profile is measured using an automatic recording spectrophotometer.
• The Tm is affected by the ionic strength of the buffer used; 0.5x SSC is a buffer commonly utilized.
• Both the unknown sample and reference DNA should be processed in the same way.
• Rinsing cuvettes with water can dilute the buffer, altering the thermal stability of the DNA.
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