Nucleic Acids Research, Vol 24, Issue 4 707-712, Copyright © 1996 by Oxford University Press
SH Ke and RM Wartell
Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine
the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent
mismatched base pairs, and eight DNAs with Watson- Crick base pairs at the
same positions. Heteroduplex DNAs containing two adjacent mismatches were
formed by melting and reannealing pairs of homologous 373 bp DNA fragments
differing by two adjacent base pairs. Product DNAs were separated based on
their thermal stability by parallel and perpendicular TGGE. The
polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the
DNA melting temperatures. The order of stability was determined in the
sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the
mismatched or Watson-Crick base pairs. The identity of the mismatched bases
and their stacking interactions influence DNA stability. Mobility
transition melting temperatures (T u) of the DNAs with adjacent mismatches
were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with
the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG),
created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same
sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol
more stable in free energy than d(AA).d(TT) base pairs. This result
confirms the unusual stability of the d(GA).d(GA) sequence previously
observed in DNA oligomers. All other DNAs with adjacent mismatched base
pairs were less stable than Watson-Crick homoduplex DNAs. Their relative
stabilities followed an order expected from previous results on single
mismatches. Two homoduplex DNAs with identical nearest neighbor sequences
but different next-nearest neighbor sequences had a small but reproducible
difference in T u value. This result indicates that sequence dependent next
neighbor stacking interactions influence DNA stability.
ARTICLES
The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA.
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