Crosslinking of PCR primers reduces unspecific amplification products in multiplex PCR

Noa Wolff; Andreas F Geiss; Ivan Barišić

doi:10.1016/j.mimet.2020.106051

Crosslinking of PCR primers reduces unspecific amplification products in multiplex PCR

J Microbiol Methods. 2020 Sep 8:178:106051. doi: 10.1016/j.mimet.2020.106051. Online ahead of print.

Authors

Noa Wolff¹, Andreas F Geiss², Ivan Barišić³

Affiliations

¹ Molecular Diagnostics, AIT Austrian Institute of Technology, Giefinggasse 4, Vienna, 1210, Austria. Electronic address: Noawolff@outlook.de.
² University of Natural Resources and Life Sciences, Muthgasse 11, Vienna, Austria.
³ Molecular Diagnostics, AIT Austrian Institute of Technology, Giefinggasse 4, Vienna, 1210, Austria.

PMID: 32911035
DOI: 10.1016/j.mimet.2020.106051

Abstract

The polymerase chain reaction is not only essential for many DNA-based diagnostic methods but is also exploited in other molecular methods that require an upstream amplification step. Multiplex PCRs are especially attractive as they reduce the number of individual reactions. However, the multiplexing efficiency is impaired by primer interactions such as the formation of primer dimers. In this study, covalent crosslinking of primers via their 5'-ends was used to avoid those undesired effects. The specificity of the primers as well as the efficiency of the PCR could be increased upon primer crosslinking in reactions containing up to 34 primer pairs targeting the most important antibiotic resistance genes in a single multiplex reaction.

Keywords: DNA-based diagnostics, antibiotic resistance genes; High-throughput detection; Multiplex PCR; Primer secondary structures.