Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries

BMC Genomics. 2019 Jul 11;20(Suppl 7):536. doi: 10.1186/s12864-019-5847-2.


Background: Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC-ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC-ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC-ROI combinations, and as a result lower the performance of the assays.

Results: To identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC-ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively.

Conclusions: We have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings.

Keywords: Barcode; Chimeric DNA molecules; Conventional PCR; Emulsion PCR (ePCR); Massively parallel reporter assay (MPRA); Next-generation sequencing.

MeSH terms

  • DNA / genetics*
  • DNA Primers
  • Gene Library*
  • High-Throughput Nucleotide Sequencing
  • Plasmids*
  • Polymerase Chain Reaction / methods*
  • Templates, Genetic


  • DNA Primers
  • DNA