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Cited by 35Molecular Biology Techniques and ApplicationsCRISPR and Genetic EngineeringGenomics and Phylogenetic Studies

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

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Omelina, Evgeniya S., Ivankin, Anton V., Letiagina, Anna E., Pindyurin, Alexey V.·2019·BMC Genomics
DOI 10.1186/s12864-019-5847-2PMC PMC6620194
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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.

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  • Emulsion PCR was used to reduce PCR bias and prevent chimeric PCR products.

    “Emulsion polymerase chain reaction (PCR) was used to recover gRNA and UCB sequences from plasmid libraries, genomic DNA extracted from lentivirus-infected hES cells and cells sorted from CHOOSE mosaic organoids as well as 10× single-cell complementary DNA libraries to reduce PCR bias and to prevent the generation of chimeric PCR products56,57.”

    From Single-cell brain organoid screening identifies developmental defects in autism (2023) · cites this paper as [56]

    The Background and Results (pages unavailable) support using optimized emulsion PCR to minimize chimeric products from MPRA plasmid libraries. The paper does not establish that it reduces PCR bias, and its results do not address the gRNA, genomic-DNA, organoid, or single-cell libraries named in the citing sentence. (LLM, conf=0.88)

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Cited by

Sorted by citations

Authors
Omelina, E. S., Ivankin, A. V., Letiagina, A. E., & Pindyurin, A. V.
Affiliations
  • Institute of Molecular and Cell Biology (RU)
  • Novosibirsk State University (RU)
Corresponding author
Pindyurin, Alexey V.
ORCID
Venue
BMC Genomics
Publisher
Springer Science and Business Media LLC
Published
2019-07
Volume
20
Issue
S7
Pages
536-536
ISSN
1471-2164 (ISSN-L)
DOI
10.1186/s12864-019-5847-2
PMC
PMC6620194
OpenAlex
W2957738448
Cited by
35
References
3
Citations by year
2026: 5 · 2025: 5 · 2024: 5 · 2023: 10 · 2022: 2 · 2021: 4 · 2020: 1 · 2019: 3
FWCI
0.83
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Genomics (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Siberian Branch, Russian Academy of Sciences
  • Russian Science Foundation (16-14-10288, 18-74-, 18-74-00050)
  • Russian Academy of Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
massively parallel reporter assay, chimeric DNA, PCR optimization, plasmid library, emulsion PCR, spacer sequences, DNA regulatory elements
MeSH terms
DNA, Plasmids, Templates, Genetic, Gene Library, Polymerase Chain Reaction, DNA Primers, High-Throughput Nucleotide Sequencing

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