Causes of bimodal melting curve:Asymmetric guaninecytosine (GC) distribution causing two peaks in melting curve and affecting their shapes
The aim of this study was to present a new situation in which a relatively single short PCR-product might show two separate peaks with sequence specific shapes at the dissociation curve. SYBR-Green I real-time RT-PCR was performed on Lhcgr-gene transcripts in rats. Different programs were used for melting curve simulation and estimating Tm. Statistical tests were performed to determine whether two peaks at the dissociation curve were belonging to a single template. A bimodal melting curve was observed in real-time RT-PCR on a short segment (169 bp) of Lhcgr gene with a single band in gel electrophoresis. Sequencing of the Cloned PCR-product was compatible with template sequence. Realtime PCR using the vector conveying interested sequence, showed again two peaks at dissociation curve. The GC-content of first 100 bases (75%) and last 69 bases (42%) were significantly different. DNA melting simulation programs also confirmed the bimodal pattern, although, their height and wideness were different to actual peaks. Due to the asymmetric GC distribution effect on dissociation curve in short sequences, it is highly recommended to use DNA melting simulation programs to predict the number of peaks in the melting curve when designating primers; however, predicted peak shapes are not always accurate.
Key words: Asymmetric GC distribution, bimodal melting curve, DNA melting simulation, SYBR-green I realtime PCR.