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

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

doi:10.3390/ijms21145119

Cited by 2 publications

(1 citation statement)

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“…Previously, we developed an oligoribonucleotide (ORN)-mediated blocking PCR named ORN interference-PCR (ORNi-PCR) (Figure A) . ORNi-PCR clearly discriminates nucleotide differences such as indel mutations (which are introduced by genome editing events), single-nucleotide mutations in cancer cells, and nucleotide polymorphisms. − In general, DNA polymerases lacking 5′–3′ exonuclease activity are used for ORNi-PCR because those retaining the 5′–3′ exonuclease activity, such as Taq DNA polymerase, can remove a hybridized ORN during DNA extension. In fact, we observed that DNA amplification by Taq DNA polymerase is not inhibited by an ORN .…”

Section: Introductionmentioning

confidence: 99%

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences

2023

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Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5′–3′ exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

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Section: Introductionmentioning

confidence: 99%

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences

2023

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Oligoribonucleotide interference-PCR: principles and applications

Shimizu

Fujita

Fujii

2022

Biology Methods and Protocols

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PCR amplification of multiple templates using common primers is used widely for molecular biological research and clinical diagnosis. However, amplifying a specific DNA sequence harboring a mutation that is present in a small number of mutant cells within a large population of normal cells (e.g., as in cancer) in a tissue is difficult using the original PCR protocol. Thus, some measures are necessary to suppress amplification of background signals. To achieve this, we developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) technology in which an ORN (short RNA) hybridizes with a complementary DNA sequence to inhibit PCR amplification across the specific target sequence. ORNs can be prepared inexpensively, and ORNi-PCR can be carried out easily by adding ORNs to the PCR reaction mixture. Suppressing amplification of target sequences by ORNi-PCR is useful for detecting target sequence mutations. We showed that ORNi-PCR can discriminate single nucleotide mutations in cancer cells and indel mutations introduced by genome editing. We also showed that ORNi-PCR can identify the CpG methylation status of a target sequence within bisulfite-treated DNA, and can enrich DNA sequences of interest from a DNA mixture by suppressing amplification of unwanted sequences. Thus, ORNi-PCR has many potential applications in various fields, including medical diagnosis and molecular biology. In this review, we outline the principles of the ORNi-PCR method and its use to detect nucleotide mutations in a variety of specimens.

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

Cited by 2 publications

References 29 publications

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences

Oligoribonucleotide interference-PCR: principles and applications

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