FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations.
Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5'-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to approximately 0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.
Amicarelli
Giulia
G
DiaSorin SpA, via Crescentino snc, 13040 Saluggia (VC), Italy.
Shehi
Erlet
E
Makrigiorgos
G Mike
GM
Adlerstein
Daniel
D
D002460:Cell Line; D003062:Codon; D015179:Colorectal Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D013997:Time Factors; D018631:ras Proteins / Q000235:genetics*
D003062:Codon; C117307:KRAS protein, human; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins