International Journal of Molecular Sciences Article Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR Takeshi Shimizu, Toshitsugu Fujita *, Sakie Fukushi, Yuri Horino and Hodaka Fujii * Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan; [email protected] (T.S.); [email protected] (S.F.); [email protected] (Y.H.) * Correspondence: [email protected] (T.F.); [email protected] (H.F.) Received: 28 May 2020; Accepted: 17 July 2020; Published: 20 July 2020 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: ORNi-PCR; PCR; acetone-fixed paraffin-embedded (AFPE); formalin-fixed paraffin-embedded (FFPE); bisulfite; CpG methylation; polymorphism; mutation; epidermal growth factor receptor (EGFR) 1. 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 specifically detect a target mutation. Alternatively, PCR can amplify a sequence across a target mutation, followed by DNA sequencing of the amplicon to confirm the presence of the mutation. Nucleotide mutations can also 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. By monitoring DNA amplification, one can determine the presence or absence of mutations in a target sequence. Previously, we developed a form of blocking PCR, called oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) [5], in which an ORN serves as the sequence-specific blocker, as shown in Figure1. ORNs can be flexibly designed and cost-effectively synthesized, representing advantages over other blockers, such as artificial nucleic acids. We showed that ORNi-PCR can discriminate differences in nucleotide sequences, such as single-nucleotide mutations in cancer cells, as well as indel mutations introduced by genome-editing [6–9]. Thus, ORNi-PCR is a useful tool for use in the biological and medical fields. Int. J. Mol. Sci. 2020, 21, 5119; doi:10.3390/ijms21145119 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 16 Int. J. Mol. Sci. 2020, 21, 5119 2 of 14 FigureFigure 1. Schematic 1. Schematic diagram diagram of ORNi-PCR. of ORNi-PCR. In ORNi-PCR,In ORNi-PCR an, an ORN ORN hybridized hybridized with with a targeta target site site blocks elongationblocks (blueelongation lines) (blue of a lines) DNA of polymerase a DNA polymerase that does that not does possess not possess 50 to 5 3′ to0 exonuclease 3′ exonuclease activity. activity.If the targetIf sitethe target is mutated site is mutated (red dots), (red thedots), ORN the ORN cannot cannot effectively effectively hybridize hybridize to to thethe target site, site, resulting resulting in targetin amplification. target amplification. In previousIn previous work, work, we usedwe used DNA DNA extracted extracted from from cultured cultured cell cell lines lines or complementary or complementary DNA DNA (cDNA) (cDNA) reverse-transcribed from freshly prepared RNA for ORNi-PCR [6–9]. Such DNA is generally reverse-transcribed from freshly prepared RNA for ORNi-PCR [6–9]. Such DNA is generally of high of high quality and can readily be used as a template for ORNi-PCR or conventional PCR. By contrast, qualityDNA and extracted can readily from befixed used tissue as specimens a template is for genera ORNi-PCRlly damaged orconventional and unsuitable PCR. for PCR, By contrast,as fixation DNA extractedprocedures from fixed using tissue formalin specimens and isorganic generally solvents damaged, such and as unsuitable alcohol and for PCR,acetone, as fixation cause proceduresDNA usingfragmentation formalin and [10–13]. organic On solvents, the othersuch hand, as althou alcoholgh bisulfite-treated and acetone, cause DNA DNAhas been fragmentation widely used for [10 –13]. On thePCR other to distinguish hand, although the state bisulfite-treated of CpG methylation, DNA th hase bisulfite been widely treatment used that for converts PCR to cytosine distinguish to the stateuracil of CpG also methylation, damages DNA the bisulfite and causes treatment difficulty that with converts PCR cytosineamplification to uracil [14,15]. also Considering damages DNA the and causespotential difficulty use with of ORNi-PCR PCR amplification for clinical diagnosis, [14,15]. Considering it is important the to potential determine use whether of ORNi-PCR such damaged for clinical diagnosis,DNA is it iscompatible important with to determineORNi-PCR. whether such damaged DNA is compatible with ORNi-PCR. In thisIn this study, study, we we conducted conducted feasibility feasibility studies studies to determine determine whether whether tissue tissue specimen specimen DNA DNA and and bisulfite-treated DNA were compatible with ORNi-PCR. We showed that ORNi-PCR could bisulfite-treated DNA were compatible with ORNi-PCR. We showed that ORNi-PCR could discriminate discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded (AFPE) nucleotidespecimens differences and formalin-fixed in DNA extracted paraffin-embedded from acetone-fixed (FFPE) specimens. paraffin-embedded In addition, (AFPE) we showed specimens that and formalin-fixedORNi-PCR paracouldffi discriminaten-embedded CpG (FFPE) methylation specimens. status In in addition, a target sequence we showed using that bisulfite-treated ORNi-PCR could discriminateDNA. Our CpG results methylation suggest the statuspotential in utility a target of ORNi-PCR sequence usingfor a variety bisulfite-treated of applications DNA. in biological Our results suggestand the medical potential fields. utility of ORNi-PCR for a variety of applications in biological and medical fields. 2. Results2. Results 2.1. ORNi-PCR2.1. ORNi-PCR Using Using AFPE AFPE or or FFPE FFPE Specimen Specimen DNADNA FormalinFormalin has beenhas been widely widely used used to preserve to preserve tissue tiss specimensue specimens for for a longa long time time because because it fixesit fixes tissues moretissues strongly more than strongly other than organic other solvents. organic solvents. However, However, organic organic solvents, solvents, including including acetone, acetone, has also has been employedalso been to fix employed tissues for to some fix tissues cases, infor which some mildercases, fixationin which was milder suitable fixation for downstreamwas suitable analyses,for downstream analyses, such as immunohistochemistry with specific antibodies [16,17]. Indeed, we such as immunohistochemistry with specific antibodies [16,17]. Indeed, we have used acetone fixation have used acetone fixation for the immunohistochemistry of rat tissues [18]. Thus, we first examined for thewhether immunohistochemistry DNA extracted from ofAFPE rat tissuestissue specim [18].ens Thus, could we be first used examined for ORNi-PCR. whether Previously, DNA extracted we fromreported AFPE tissuea nucleotide specimens polymorphism could be in usedthe rat for Gstm1 ORNi-PCR. gene (encoding Previously, glutathione we S-transferases reported a nucleotidemu 1); polymorphismSprague–Dawley in the (SD) rat Gstm1 rats havegene (encodingthe wild-type glutathione (WT) sequence S-transferases (198Lys (AAG)- mu 1);199 Sprague–DawleySer (AGC)) (SD) rats have the wild-type (WT) sequence (198Lys (AAG)-199Ser (AGC)) (considering nucleotides G and A, we hereafter call this type of nucleotides as “GA-type”), whereas Hirosaki hairless rats have a nucleotide polymorphism (198Asn (AAT)-199Cys (TGC)) (considering nucleotides T and T, we hereafter call this type of nucleotides as “TT-type”), as shown in Figure2A[ 19]. In this experiment, we investigated whether ORNi-PCR could discriminate the polymorphism in DNA extracted from Int. J. Mol. Sci. 2020, 21, 5119 3 of 14 AFPE rat liver specimens. To this end, we designed ORN_GA2 and ORN_GA3, 20-base and 22-base ORNs, complementary to the GA-type sequence, respectively, as shown in Figure2A. DNA was extracted from sliced AFPE rat liver specimens and used for ORNi-PCR, as described previously [7].