Increased single-nucleotide discrimination of PCR by primer probes bearing hydrophobic 4'C modifications.
We report on significantly increased selectivity of real-time PCR through employment of primer probes that bear hydrophobic 4'C modifications at the 3'-terminal nucleotide. The primer probes were designed to bind the target sequences in such a way that the 3'-terminal nucleotide defines whether a matched or a single mismatched basepair is present depending on the respective target sequence. Several commercially available thermostable DNA polymerases belonging to different DNA polymerase families were tested for their efficacy in discriminating between PCR amplification of matched substrates and duplexes that contain a single mismatch. It turned out that, depending on the 4'C modification and the employed DNA polymerase, significantly increased differentiation between single matches and mismatches could be observed with real-time PCR. The degrees of the observed effects varied with the employed 4'C modification and the sequence context studied. The system is robust enough to work faithfully under several buffer conditions. Our approach should be useful for the direct diagnosis of single nucleotide variations within genes, like single nucleotide polymorphisms or mutations, by PCR without the need for further time- and cost-intensive post-PCR analysis.
Strerath
Michael
M
Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
Gaster
Jens
J
Summerer
Daniel
D
Marx
Andreas
A
D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry / Q000592:standards*; D004259:DNA-Directed DNA Polymerase / Q000592:standards; D015195:Drug Design; D057927:Hydrophobic and Hydrophilic Interactions; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase