Figure 1.

SYBR Green based Real-Time PCR of loxP containing sequences yields an aberrant melting curve. A Strategy for primer placement: Primers specific for the 2lox allele are placed within the loxP flanked genomic region and primers specific for the 1lox allele are placed into unique genomic sequences surrounding the remaining loxP site of the truncated allele. B Representative melting curve analysis of the 1lox reaction (the figure shows different primer concentrations measured in triplicates). This reaction consistently produced a double peak possibly due to the palindromic loxP sequence that can promote annealing of sense/sense and antisense/antisense homodimers. The same was observed when using alternative primer pairs, different annealing temperatures, different DNA concentrations and even the gel purified 1lox PCR product. C Gel electrophoresis of the 1lox Real-Time PCR reaction showed a single band, confirming that the double peak in the melting curve is not caused by the presence of an additional nonspecific amplicon.