Detection of genome-edited cells by oligoribonucleotide interference-PCR

Fujita, Toshitsugu; Yuno, Miyuki; Kitaura, Fusako; Fujii, Hodaka

doi:10.1093/dnares/dsy012

Cited by 8 publications

(18 citation statements)

References 28 publications

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“…In the third step (Step III), the optimal ORN concentration is determined. Two-Step ORNi-PCR is performed at an ORN concentration of 0.5–2 μM, since this is known to be an effective concentration range 4 . In the fourth step (Step IV), Two-Step ORNi-PCR is performed using the established optimal conditions for a given application, such as detection of nucleotide differences (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1B ). In this regard, the positions of nucleotide mismatches in the hybridised DNA/ORN are not limited to the centre of the ORN 4 . The established protocol would be straightforward but may be time-consuming to find the most optimized condition.…”

Section: Resultsmentioning

confidence: 99%

“…ORNi-PCR can be applied to detect nucleotide differences, such as insertion/deletion (indel) mutations introduced during genome editing, mutations causing diseases and single-nucleotide polymorphisms (SNPs; Fig. 1B ) 4 . Because chemical synthesis of ORNs is inexpensive, it is affordable to screen a large number of cells possessing nucleotide differences using this method.…”

Section: Introductionmentioning

confidence: 99%

“…Because chemical synthesis of ORNs is inexpensive, it is affordable to screen a large number of cells possessing nucleotide differences using this method. ORNi-PCR can be performed on end-point levels as well as real-time levels using appropriate PCR machines 4 . Thus, ORNi-PCR is a useful method, but technical improvement could allow more precise discrimination of nucleotide differences in an ORN-specific manner for applications in molecular biology and medical fields 4 .…”

Section: Introductionmentioning

confidence: 99%

“…ORNi-PCR can be performed on end-point levels as well as real-time levels using appropriate PCR machines 4 . Thus, ORNi-PCR is a useful method, but technical improvement could allow more precise discrimination of nucleotide differences in an ORN-specific manner for applications in molecular biology and medical fields 4 . In this regard, trial and error steps would still be necessary to find an optimal experimental condition for an ORN.…”

Section: Introductionmentioning

confidence: 99%

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A refined two-step oligoribonucleotide interference-PCR method for precise discrimination of nucleotide differences

Fujita

Yuno

Kitaura

et al. 2018

Sci Rep

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We previously developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which an ORN hybridises with a complementary DNA sequence and inhibits PCR amplification across the sequence in a sequence-specific manner. Suppression of target amplification by ORNi-PCR can be used to detect nucleotide differences such as mutations in a target sequence. In the present study, we refined the ORNi-PCR method and established a detailed technical protocol to precisely discriminate single-nucleotide differences. We first revealed that a two-step (denaturing and annealing plus elongation) rather than a standard three-step (denaturing, annealing and elongation) method is more suitable for stably hybridising an ORN to its target DNA sequence for sequence-specific suppression of target amplification. We then optimised the ORNi-PCR method using two-step cycles and established a step-by-step technical protocol. The optimised Two-Step ORNi-PCR method could discriminate single-nucleotide differences in genomic DNA or cDNA introduced by genome editing or mutations in cancer cells. In addition, we showed that Two-Step ORNi-PCR can detect the cancer cells possessing a single nucleotide mutation in a target locus mixed with a large number of cells harboring wild-type sequences in the locus so that the number of the cancer cells is only 0.2% of the total cell number. Two-Step ORNi-PCR is useful for simple, precise, cost-effective and positive detection of nucleotide differences in a wide range of molecular biology and medical applications.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A refined two-step oligoribonucleotide interference-PCR method for precise discrimination of nucleotide differences

Fujita

Yuno

Kitaura

et al. 2018

Sci Rep

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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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Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

2021

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Isothermal DNA amplification, such as recombinase polymerase amplification (RPA), is well suited for point-of-care testing (POCT) as it does not require lengthy thermal cycling. By exploiting DNA amplification at low temperatures that do not denature heat-sensitive molecules such as proteins, we have developed a blocking RPA method to detect gene mutations and examine the epigenetic status of DNA. We found that both nucleic acid blockers and nuclease-dead clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins suppress RPA reactions by blocking elongation by DNA polymerases in a sequence-specific manner. By examining these suppression events, we are able to discriminate single-nucleotide mutations in cancer cells and evaluate genome-editing events. Methyl-CpG binding proteins similarly inhibit elongation by DNA polymerases on CpG-methylated template DNA in our RPA reactions, allowing for the detection of methylated CpG islands. Thus, the use of heat-sensitive molecules such as proteins and ribonucleoprotein complexes as blockers in low-temperature isothermal DNA amplification reactions markedly expands the utility and application of these methods.

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Detection of genome-edited cells by oligoribonucleotide interference-PCR

Cited by 8 publications

References 28 publications

A refined two-step oligoribonucleotide interference-PCR method for precise discrimination of nucleotide differences

A refined two-step oligoribonucleotide interference-PCR method for precise discrimination of nucleotide differences

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

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

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