title: TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes.
abstract: Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ~10-fold increase in the detection sensitivity for mutantBRAFandNRASgenes was achieved.
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Botezatu
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Irina O
authors.initials: IO
authors.lastname: Panchuk
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Stroganova
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Anastasia I
authors.initials: AI
authors.lastname: Senderovich
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Valentina N
authors.initials: VN
authors.lastname: Kondratova
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Valery P
authors.initials: VP
authors.lastname: Shelepov
authors.affiliation: N.N. Blokhin Cancer Research Center, Moscow, Russia.
authors.forename: Anatoly V
authors.initials: AV
authors.lastname: Lichtenstein
mesh_terms: D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry*; D020558:GTP Phosphohydrolases / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D015342:DNA Probes; D008565:Membrane Proteins; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human
keywords: BRAF; DNA melting analysis; NRAS; TaqMan probes; blocking agents; mutation detection