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

Evgeniya S. Omelina, Anton V. Ivankin, Anna E. Letiagina, Alexey V. Pindyurin

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number536
Pages (from-to)536
Number of pages10
JournalBMC Genomics
Volume20
Issue numberSuppl 7
DOIs
Publication statusPublished - 11 Jul 2019

Keywords

  • Barcode
  • Chimeric DNA molecules
  • Conventional PCR
  • Emulsion PCR (ePCR)
  • Massively parallel reporter assay (MPRA)
  • Next-generation sequencing
  • SEQUENCES
  • CIS-REGULATORY FUNCTION
  • COAMPLIFICATION
  • IDENTIFICATION
  • RIBOSOMAL-RNA GENES
  • AMPLIFICATION
  • DISSECTION
  • MEDIATED RECOMBINATION
  • CONSEQUENCE
  • FREQUENCY

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