**6. Discussion**

The main aim of our research was to determine the sequences of chosen P- and F17-fimbriae genes among *E. coli* isolated from faecal samples of diarrhoeic dogs. As we assumed that the fimbriae of such *E. coli* strains, due to already known variations of P- and F17-fimbriae, might have nucleotide differences, the annealing temperature of 55°C in the PCRs was used. To our surprise, even though only PCR products of expected sizes were cloned, many of the obtained PCR clones, in the case of PCR products obtained with *papA* primers 75% and in the case of PCR product obtained with *F17G* primers 50%, carried false inserts. Nucleotide sequence analysis revealed that also in the case of *papEF* clones, even though the cloned inserts were as hoped for fimbrial inserts, even if they were Prs-fimbrial genes, the binding site of the reverse primer was not the expected one. The high percentages of false PCR products were obtained when PCR primers with a high melting temperature (Tm) were used at the annealing temperature of 55°C—primer 22 has the Tm of 84.1°C, and 75% of false PCR products were obtained with this primer; primers F17G-1 and F17G-2 have the Tm of 72.4°C and 76.4°C, respectively, and 50% of false PCR products were obtained with them. In the consecutive PCR amplifications with the primers 22 and 23, the annealing temperature was raised to 60°C, and from these PCRs more PCR products were obtained, namely, 16. All 16 were cloned and analysed, and all clones were with correct inserts (data not shown).

### **7. Conclusion**

To conclude, we all know that with PCR, we can obtain false unspecific products, and we believe that such PCR products will be distinguished from right PCR products, because the false PCR products will not be of the correct expected size; however, our results showed that also PCR products of the expected size can be false PCR products. In order to avoid false positive PCR results, it is therefore essential to use the right annealing temperature that should not be too different from the primer's melting temperature.

#### **Acknowledgements**

The author is very thankful to Wim Gaastra for the primer nucleotide sequences. This analysis was supported by the Slovenian Research Agency (P1-0198).

**177**

**Author details**

Marjanca Starčič Erjavec

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia

\*Address all correspondence to: marjanca.starcic.erjavec@bf.uni-lj.si

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

*DOI: http://dx.doi.org/10.5772/intechopen.85164*

#### **Conflict of interest**

The author has no conflict of interest.

*Annealing Temperature of 55°C and Specificity of Primer Binding in PCR Reactions DOI: http://dx.doi.org/10.5772/intechopen.85164*
