Influence of Unequal Amplification of Methylated and Non-Methylated Template on Performance of Pyrosequencing

Olga Taryma-Lesniak, Tina E. Kjeldsen, Lise Lotte Hansen, Tomasz K. Wojdacz*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

1 Citation (Scopus)

Abstract

Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

Original languageEnglish
Article number1418
JournalGenes
Volume13
Issue8
ISSN2073-4425
DOIs
Publication statusPublished - Aug 2022

Keywords

  • DNA methylation
  • in vitro diagnostic
  • IVD
  • pyrosequencing

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