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

Takeshi Shimizu,Yuri Horino,Sakie Fukushi,Toshitsugu Fujita,Hodaka Fujii

doi:10.3390/ijms21145119

Takeshi Shimizu, Yuri Horino + Show 3 more

Open Access

https://doi.org/10.3390/ijms21145119

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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.

Highlights

PCR is widely used throughout biology and medicine [1,2], and PCR-based detection of nucleotide mutations has been used in clinical diagnoses of intractable diseases, such as cancer [3]
We showed that ORNi-PCR could discriminate discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded (AFPE)
We first examined whether DNA extracted whether DNA extracted from AFPE tissue specimens could be used for ORNi-PCR

Summary

Introduction

PCR is widely used throughout biology and medicine [1,2], and PCR-based detection of nucleotide mutations has been used in clinical diagnoses of intractable diseases, such as cancer [3]. In this context, PCR with a specific primer set can detect a target mutation. Nucleotide mutations can be detected by blocking PCR [4], in which a blocker oligonucleotide hybridized to a target sequence suppresses PCR amplification across the target sequence in a sequence-specific manner. If the target sequence is mutated, hybridization is incomplete, and PCR amplification would not be suppressed. We developed a form of blocking PCR, called oligoribonucleotide (ORN)

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