Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR

Int J Mol Sci. 2020 Jul 20;21(14):5119. doi: 10.3390/ijms21145119.

Abstract

Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.

Keywords: CpG methylation; ORNi-PCR; PCR; acetone-fixed paraffin-embedded (AFPE); bisulfite; epidermal growth factor receptor (EGFR); formalin-fixed paraffin-embedded (FFPE); mutation; polymorphism.

MeSH terms

  • Acetone / chemistry
  • Animals
  • Cell Line
  • CpG Islands*
  • DNA Methylation*
  • Formaldehyde / chemistry
  • Humans
  • Liver / metabolism
  • Oligoribonucleotides / genetics
  • Paraffin Embedding
  • Polymerase Chain Reaction / methods*
  • Rats
  • Rats, Sprague-Dawley
  • Sulfites / chemistry
  • Tissue Fixation

Substances

  • Oligoribonucleotides
  • Sulfites
  • Acetone
  • Formaldehyde
  • hydrogen sulfite