RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes

Hou, Jingli; Liu, Xipeng; Pei, Dongli; Liu, Jianhua

doi:10.1016/j.bbrc.2007.03.075

Cited by 12 publications

(14 citation statements)

References 21 publications

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“…Outside positions "-3 to +1", the presence of a mismatch had little or no effect on cleavage by the enzyme. As has been seen with other RNase H2 enzymes [31,32], the greatest effect was observed for mismatches flanking the cleavage site (positions "-1" and "0"). A mismatch at these locations inhibited cleavage by approximately 10-fold.…”

Section: Resultssupporting

confidence: 68%

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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: Resultssupporting

confidence: 68%

“…Consistent with its role in DNA repair, Type II RNase H enzymes are also able to cleave substrates where there is an RNA:DNA base pair mismatch, but at a rate reduced compared to the corresponding perfect duplex [31,32,46-48]. For P.a .…”

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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“…[24–25,34] incorporated single ribonucleotides into the loop portion of their MB design with the intention that RNase H nickases would nick the hybridized probes in what amounts to a very useful assay for the detection of single nucleotide polymorphisms (SNPs) [34]. One major difference between the approach presented in this study and that of the Liu lab is that the SNP probe relies principally on DNA-DNA hybridization for target recognition and stem melting.…”

Section: Discussionmentioning

confidence: 99%

“…Technically, previous studies performed by the Liu lab [24–25,34] have employed CpRNase HII [24–25] and TthRNase HII [34] to selectively nick MBs containing a single ribonucleotide in their the loop sequence (DNA-rN1-DNA MBs) when bound to target DNA with a perfectly matched sequence specifically nicked the MB loop portion of the target•MB helix, allowing the liberated target to bind to another free and unprocessed probe. This amounted to an ingenious assay for detecting single-nucleotide polymorphisms (SNPs) that takes advantage of CPT to amplify the signal when an SNP is present.…”

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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“…Two of type 2 RNases H from Chlamydia pneumoniae, Rnase hii (cpRnase hii) and RNase HIII (CpRNase HIII) were successfully expressed and purified in our lab previously (17). Furthermore, our research demonstrated that cpRnase hii could cleave DnA-rn 1 -DnA/ DNA duplex effectively and the cleavage rates of CpRNase HII were negatively affected by mismatches carried by the DnA-rn 1 -DNA/DNA duplex (5).…”

Section: Introductionmentioning

confidence: 87%

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

Hou

Liu

2012

Biotechnology & Biotechnological Equipment

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RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes

Cited by 12 publications

References 21 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

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

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

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