SNP analysis using catacleave probes

Harvey, John; Brant, Steven R.; Knutson, Jay R.; Han, Mira

doi:10.1002/jcla.20240

Cited by 8 publications

(5 citation statements)

References 40 publications

(31 reference statements)

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“…Several groups have employed RNase H in SNP discrimination assays using unbiased amplification of the target sequence linked to cleavage of an RNA-containing probe. Harvey, Han, and colleagues described the use of a thermostable RNase H1 enzyme in genotyping assays where cleavage of a fluorescence-quenched probe having four sequential RNA bases was used to discriminate base identity [ 47 ]. The assay was coupled to PCR and cleavage of the probe occurred in real time during thermocycling, as in a 5'-nuclease assay.…”

Section: Discussionmentioning

confidence: 99%

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

et al. 2011

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BackgroundThe polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences.ResultsPyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair.ConclusionrhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

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Section: Discussionmentioning

confidence: 99%

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

et al. 2011

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“…To verify the proof of concept of the assay, equimolar amounts (50 nM) of both dye‐labeled DNA probes, which flanked the point mutation in the target RNA, were mixed for 10 min in a Tris‐buffered solution with matched RNA, and ligated by T4 DNA ligase . The DNA–RNA hybrids were digested by RNase H for removal of the RNA template . The emission fluorescence spectrum of each resulting ligated sample for quantitation of the matched RNA was scanned using a fluorimeter upon excitation at 550 nm.…”

Section: Resultsmentioning

confidence: 99%

Allele‐specific, Hybridization‐based, Washing‐free Fluorescence Signal Production Platforms for Quantitation of Single‐Base Change (C → U) in RNA

Park

Hah

2015

Bulletin Korean Chem Soc

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A single nucleotide polymorphism (SNP) is a single base-pair substitution that commonly constitutes the genetic variation in individuals. Because of its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we report four types of washing-free (i.e., separation-free or sequential addition of reagents) and polymerase chain reaction (PCR)-free fluorescence signal production platforms for quantitative detection of a single-base mismatch in RNA and compare their analytical efficiency. The RNA-templated SNP detection methods in this study are based on the allelespecific hybridization approach and/or on the allele-specific oligonucleotide ligation approach, and have been successfully applied to the quantitation of single-base mutation (C ! U) in RNA of the BCR-ABL fusion gene, by endpoint measurements of fluorescence intensity. These methods have several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cells, no requirement of PCR amplification, performance of homogeneous detection, easy design of detection probes, and suitability for large-scale genotyping.

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“…In comparison to one of its closest relatives found in the literature, the CataCleave probe [7–8], the R/D-MB + thermostable RNase H system offers several advantages due to its nature as a MB. For example, hybridization of the CataCleave probe to its target sequence does not lead to any change in donor fluorescence emission, meaning that detection requires that RNase H mediated cleavage is applied.…”

Section: Discussionmentioning

confidence: 99%

“…It is seen from Eqn. 1 that the S:N ratio of a particular MB design may be attenuated by: 1) incomplete initial quenching in the absence of targets, which increases F close ; 2) incomplete abolishment of FRET once a target is bound, which decreases F open ; and 3) a lack of the type of processive cycling seen in the Catacleave probe [7–8] or the SNP assay of the Liu lab [34] which would enable one target molecule to react with multiple MBs and reduce the concentration of targets needed to achieve a particular F open . Most attempts at improving the S:N ratio have focused on improving the MB design to either reduce F close by enhancing the initial quenching efficiency or increase F open by enhancing the performance of the donor [5].…”

Section: Introductionmentioning

confidence: 99%

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

Jacroux

Rieck

Chen

et al. 2013

Analytical Biochemistry

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A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. Addition of thermostable RNase H resulted in the cleavage of the RNA loop which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9x above background), resulting in a ~2–2.8 fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time PCR reactions by measuring enhancement of donor fluorescence upon R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods.

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SNP analysis using catacleave probes

Cited by 8 publications

References 40 publications

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

Allele‐specific, Hybridization‐based, Washing‐free Fluorescence Signal Production Platforms for Quantitation of Single‐Base Change (C → U) in RNA

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

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