Biophysical Chemistry, 2005, Vol. 117, No. 3, pp 207-215.
Melting temperatures of nucleic acids: Discrepancies in analysis
Richard Owczarzy
Reprint
Melting temperature, Tm, is an important property of
nucleic acid
duplexes. It is typically determined from spectroscopic or calorimetric melting
experiments. More than one analytical method has been used to extract
Tm values
from experimental melting data. Unfortunately, different methods do not give
the same results; the same melting data can be assigned different
Tm values
depending upon which method is used to process that data. Inconsistencies or
systematic errors between Tms reported in published data sets
can be
significant and add confusion to the field. Errors introduced from analysis can
be greater than experimental errors, ranging from a fraction of degree to
several degrees. Of the various methods, the most consistent and meaningful
approach defines melting temperature as the temperature at the transition
midpoint where half of the base pairs are melted and standard free energy is
zero. Assuming a two-state melting behavior, we present here a set of general
equations that can be used to reconcile these analytical Tm
differences and
convert results to the correct melting temperatures at the transition midpoint.
Melting temperatures collected from published sources, which were analyzed
using different methods, can now be corrected for these discrepancies and
compared on equal footing. The similar corrections apply to Tm
differences
between calorimetric and spectroscopic melting curves. New algorithm for
selection of linear sloping baselines, 2nd derivative method, is suggested,
which can be used to automate melting curve analysis.
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