Nucleic Acids Research Advance Access originally published online on October 11, 2007
Nucleic Acids Research 2007 35(19):e131; doi:10.1093/nar/gkm809
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Nucleic Acids Research, 2007, Vol. 35, No. 19 e131
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations
1DiaSorin SpA, via Crescentino snc, 13040 Saluggia (VC), Italy and 2Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA
* To whom correspondence should be addressed. Tel: +0039 0331581473; Fax: +0039 03311547; Email: daniel.adlerstein{at}diasorin.it
Received June 27, 2007. Revised September 17, 2007. Accepted September 18, 2007.
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 
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.
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