title: Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations.
abstract: The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5-3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity-all of the qualities required for clinical genotyping.
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Botezatu
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia.
authors.forename: Irina O
authors.initials: IO
authors.lastname: Nechaeva
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia.
authors.forename: Аnna М
authors.initials: АМ
authors.lastname: Stroganova
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia.
authors.forename: Anastasia I
authors.initials: AI
authors.lastname: Senderovich
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia.
authors.forename: Valentina N
authors.initials: VN
authors.lastname: Kondratova
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia.
authors.forename: Valery P
authors.initials: VP
authors.lastname: Shelepov
authors.affiliation: N. N. Blokhin Russian Cancer Research Center, 115478 Moscow, Russia. Electronic address: alicht@mail.ru.
authors.forename: Anatoly V
authors.initials: AV
authors.lastname: Lichtenstein
mesh_terms: D000483:Alleles; D001483:Base Sequence; D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000473:pathology; D016612:Paraffin Embedding; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D044366:Transition Temperature; D018631:ras Proteins / Q000235:genetics
chemical_list: D015342:DNA Probes; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D004247:DNA; D048493:Proto-Oncogene Proteins B-raf; D018631:ras Proteins
keywords: BRAF; DNA melting analysis; KRAS; Mutation scanning; NRAS; TaqMan probes