RESUMO
A number of probe systems exist for the real-time detection of PCR products. Scorpions are a unique method wherein primer and probe are combined in a single oligonucleotide. During the PCR, the probe element becomes linked directly to its complementary target site with beneficial consequences. In particular, the unimolecular mechanism of probe/target hybridization ensures rapid, reliable, and robust probing of a chosen amplicon. We discuss the design and use of Scorpions and compare their use with similar systems.
Assuntos
Primers do DNA/química , Primers do DNA/genética , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , Proteína BRCA1/genética , Transferência de Energia , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
BACKGROUND: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS: We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.
Assuntos
Biomarcadores Tumorais/genética , Fosfatidilinositol 3-Quinases/genética , Neoplasias da Mama/genética , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Melanoma/genética , Mutação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Scorpion probes, specific DNA probe sequences maintained in a hairpin-loop, can be modified to carry the components of an exciplex for use as a novel fluorescence-based method for specific detection of DNA. The exciplex partners (5'-pyrenyl and 3'-naphthalenyl) were attached to oligonucleotides via phosphoramidate links to terminal phosphate groups. Hybridization of the probe to a complementary target in a buffer containing trifluoroethanol produced an obvious fluorescence change from blue (pyrene locally excited state emission) to green (exciplex emission).
Assuntos
Sondas de DNA/metabolismo , Sondas de Oligonucleotídeos/metabolismo , Corantes Fluorescentes , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Espectrometria de FluorescênciaRESUMO
The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.
