Oligoribonucleotide (ORN) Interference-PCR (ORNi-PCR): A Simple Method for Suppressing PCR Amplification of Specific DNA Sequences Using ORNs

Tanigawa, Naoki; Fujita, Toshitsugu; Fujii, Hodaka

doi:10.1371/journal.pone.0113345

Cited by 12 publications

(20 citation statements)

References 15 publications

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“…Next, we tested other ORNs. ORNi-PCR with ORN_24b or ORN_Target, but not ORN_302F(NC), another irrelevant 21-base ORN, 15 yielded PCR patterns identical to those of ORNi-PCR with ORN_20b (Fig. 4C and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 83%

“…Lower concentrations of ORN_20b (0.1 or 0.5 μM) and ORN_Target (0.5 µM) were less effective in this respect. In contrast, ORN_306F(NC), a 25-base ORN hybridizing with an irrelevant locus (human IRF-1 locus), 15 did not affect amplification (Fig. 2B).…”

Section: Resultsmentioning

confidence: 91%

“…2A). Because DNA polymerases that retain 3′–5′ exonuclease activity, but not those that retain 5′–3′ exonuclease activity, can be utilized for ORNi-PCR, 15 we used the KOD DNA polymerase 19 to amplify a 0.9 kb region surrounding the target site (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…The Pfu DNA polymerase retains 3′–5′ exonuclease activity 19 and can be used for ORNi-PCR 15 . We therefore examined whether Pfu DNA polymerase could also be used to detect genome-edited cells.…”

Section: Resultsmentioning

confidence: 99%

“…1A). 15 In ORNi-PCR, an ORN [not an oligodeoxyribonucleotide (ODN)] with a length of 17–29 bases inhibits PCR amplification of a target DNA sequence that contains the DNA sequence complementary to the ORN (Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

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Detection of genome-edited cells by oligoribonucleotide interference-PCR

et al. 2018

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Genome editing by engineered sequence-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for analysis of gene functions. Several techniques have been developed for detection of genome-edited cells, but simple, cost-effective, and positive detection methods remain limited. Recently, we developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which hybridization of an ORN with a complementary DNA sequence inhibits amplification across the sequence. Here, we investigated whether ORNi-PCR can be used to detect genome-edited cells. First, we showed that ORNs that hybridize to a CRISPR target site in the THYN1 locus inhibited amplification across the target site, but no longer inhibited amplification after the target site was edited, resulting in mismatches. Importantly, ORNi-PCR could distinguish even single-nucleotide differences. These features of ORNi-PCR enabled detection of genome-edited cells by positive PCR amplification. In addition, ORNi-PCR was successful in discriminating genome-edited cells from wild-type cells, and multiplex ORNi-PCR simultaneously detected indel mutations at multiple loci. However, endpoint ORNi-PCR may not be able to distinguish between mono- and bi-allelic mutations, which may limit its utility. Taken together, these results demonstrate the potential utility of ORNi-PCR for the screening of genome-edited cells.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection of genome-edited cells by oligoribonucleotide interference-PCR

et al. 2018

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Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers

Liu,

Li,

Zhang

et al. 2023

Anal Bioanal Chem

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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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Oligoribonucleotide (ORN) Interference-PCR (ORNi-PCR): A Simple Method for Suppressing PCR Amplification of Specific DNA Sequences Using ORNs

Cited by 12 publications

References 15 publications

Detection of genome-edited cells by oligoribonucleotide interference-PCR

Detection of genome-edited cells by oligoribonucleotide interference-PCR

Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers

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

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