Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions

In our study, we used PCR to clone papA , papEF , papG and F17G genes of Escherichia coli isolated from faecal samples of dogs with diarrhoea. Annealing temperature of 55°C was used in the PCR. Nucleotide sequence analysis of 26 cloned PCR products showed that in PCRs with papA primers, six out of eight obtained PCR products were false due to non-specific binding of the forward primer on both DNA strands; in PCRs with the papEF primers, all seven obtained PCR products originated from specific binding of the forward primer on the 3 ′ → 5 ′ DNA strand and non-specific binding of the forward primed on the 5 ′ → 3 ′ DNA strand; and in PCRs with the F17G primers, four out of eight obtained PCR products were false due to non-specific binding of forward and reverse primer. The anticipated annealing sites for non-specific primer binding in analysed nucleotide sequences are presented. In the case of PCR products obtained with papG -specific primers, all PCR products were amplifications of the papG sequence. When the annealing temperature of papA PCRs was raised to 60°C, all obtained PCR products were amplifications of the correct DNA sequences.