title: RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  Pyrococcus 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.
  
  
  CONCLUSIONS
  rhPCR 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.
authors.affiliation: Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 5224, USA.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Dobosy
authors.forename: Scott D
authors.initials: SD
authors.lastname: Rose
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Beltz
authors.forename: Susan M
authors.initials: SM
authors.lastname: Rupp
authors.forename: Kristy M
authors.initials: KM
authors.lastname: Powers
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Walder
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D043903:Pyrococcus abyssi / Q000201:enzymology; D016914:Ribonuclease H / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D016914:Ribonuclease H