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 | This user has publicly declared that they have a conflict of interest regarding the Wikipedia article ''RNase H-dependent PCR''. |
I am a Scientist working on RNase HII at Integrated DNA Technologies. I have been involved in developing the rhPCR system, a peer-reviewed system to detect SNPs and prevent primer dimers. This work has been commented on and cited in other locations in Wikipedia already. However, I wish to reveal my potential Conflict of Interest due to my previous published work in this field.
Notes related to your Wikipedia work and activities
I intend to create a page dedicated to the rhPCR system. A draft is below:
RNase H-dependent PCR From Wikipedia, the free encyclopedia RNase H-dependent PCR (rhPCR) is a modification of the standard PCR(ref) technique. In rhPCR, the primers are designed with a removable amplification block on the 3’ end. Amplification of the blocked primer is dependent on the enzymatic activity of a thermophilic RNase HII enzyme during hybridization to the complementary target sequence. This enzyme possesses two useful characteristics that enhance the PCR. First, it has very little enzymatic activity at low temperature, enabling a “hot-start” effect. Second, the cleavage efficiency of the enzyme is reduced in the presence of mismatches near the RNA residue. This allows for reduced primer-dimer formation, and the ability to detect single-nucleotide polymorphisms. Contents • 1 Principle • 2 Applications • 3 See also • 4 Notes and references • 5 External links Principle Figure 1: rhPCR mechanism rhPCR primers consist of three sections. 1) The 5’ DNA section, equivalent in length and Tm requirements to a standard PCR primer, is extended after cleavage by the RNase HII enzyme. 2) A single RNA base provides the cleavage site for the RNase HII. 3) A short 3’ extension of four or five bases followed by a blocker (usually a short, non-extendable molecule like a propanediol) prevents extension by a DNA polymerase until removal. While free in solution, these primers are not deblocked by the RNase HII enzyme, as they must be in an RNA:DNA heteroduplex with the template in order to be cleaved. They bind to the template (Figure 1), and are cleaved by the thermostable RNase HII enzyme. This removes the block, allowing for the DNA polymerase to extend off of the primers. The cycling of the PCR reaction continues the process. rhPCR primers are designed so that after cleavage by the RNase H2 enzyme, the Tm of the primers are still greater than the annealing temperature of PCR reaction. These primers can be used in both 5’ nuclease (Taqman) and SYBR Green type of quantitative PCR. Applications rhPCR can be used for quantitative PCR and medical or environmental laboratories: • Gene expression assays • Alternative splicing(REF) • SNP genotyping • Multiplex PCR See also • Quantitative PCR • Taqman • SYBR Green • Reverse transcription polymerase chain reaction • Molecular beacon • Gene Expression
Dobosy JR, Rose SD, Beltz KR, Rupp SM, Powers KM, Behlke MA, Walder JA (August 2011). "RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers". BMC Biotechnology. 11: 80. PMC 3224242 . PMID 21831278. doi:10.1186/1472-6750-11-80.