TY - JOUR

T1 - Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries

AU - Omelina, Evgeniya S.

AU - Ivankin, Anton V.

AU - Letiagina, Anna E.

AU - Pindyurin, Alexey V.

N1 - Publisher Copyright: © 2019 The Author(s).

PY - 2019/7/11

Y1 - 2019/7/11

N2 - 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.

AB - 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.

KW - Barcode

KW - Chimeric DNA molecules

KW - Conventional PCR

KW - Emulsion PCR (ePCR)

KW - Massively parallel reporter assay (MPRA)

KW - Next-generation sequencing

KW - SEQUENCES

KW - CIS-REGULATORY FUNCTION

KW - COAMPLIFICATION

KW - IDENTIFICATION

KW - RIBOSOMAL-RNA GENES

KW - AMPLIFICATION

KW - DISSECTION

KW - MEDIATED RECOMBINATION

KW - CONSEQUENCE

KW - FREQUENCY

UR - http://www.scopus.com/inward/record.url?scp=85069044029&partnerID=8YFLogxK

U2 - 10.1186/s12864-019-5847-2

DO - 10.1186/s12864-019-5847-2

M3 - Article

C2 - 31291895

AN - SCOPUS:85069044029

VL - 20

SP - 536

JO - BMC Genomics

JF - BMC Genomics

SN - 1471-2164

IS - Suppl 7

M1 - 536

ER -