Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.

摘要: Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific mismatches effect stability long fragment. Homologous 373 bp differing substitutions in their first melting domain were employed. Heteroduplexes formed and reannealing pairs DNAs, one which 32P-labeled on its 5'-end. Product DNAs separated based parallel perpendicular temperature-gradient electrophoresis. order determined all common mismatched bases four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), d(TXT).d(AYA) with X,Y = A, T, C, or G. containing mismatch destabilized 1 5 degrees C respect homologous complete Watson-Crick pairing. Both at site neighboring stacking interactions influence destabilization caused mismatch. G.T, G.G G.A always among most stable environments examined. Purine.purine generally more than pyrimidine.pyrimidine mispairs. Our results are very good agreement data where available from solution studies short oligomers.