RESUMO
The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.
Assuntos
Fusão Gênica Artificial/métodos , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição/genética , Sequência de Bases , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 17 , Primers do DNA/química , DNA de Neoplasias/análise , Humanos , Leucemia Promielocítica Aguda/patologia , Dados de Sequência Molecular , Proteína da Leucemia Promielocítica , Translocação Genética , Proteínas Supressoras de TumorRESUMO
A microvolume fluorimeter integrated with a rapid thermal cycler allows both amplification and point mutation detection from genomic DNA in approximately 30 min. This homogeneous method combines rapid cycle DNA amplification with allele-specific fluorescent probe melting profiles for product genotyping. The amplification reaction includes a primer internally labeled with Cy5 and a 3'-fluorescein-labeled probe that spans the region of interest. During asymmetric amplification, the probe hybridizes to excess Cy5-labeled strand and is observed as fluorescence resonance energy transfer. Resonance energy transfer increases each cycle as product accumulates during amplification. When fluorescence is monitored as the temperature increases through the Tm of the probe/product duplex, a characteristic melting profile for each genotype is obtained. Fluorescence genotyping of the common C677T base substitution in the methylenetetrahydrofolate reductase gene in 110 DNA samples correlated perfectly with genotyping by restriction enzyme digestion and gel electrophoresis. The relatively stable G:T mismatch of this example gave a 3 degrees C difference in Tm from complete Watson-Crick pairing, suggesting that this homogeneous fluorescence method can be used for all single-base mismatches.
Assuntos
Genes , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Alelos , DNA/química , DNA/genética , Transferência de Energia , Corantes Fluorescentes , Frequência do Gene , Genótipo , Heterozigoto , Homozigoto , Temperatura Alta , Metilenotetra-Hidrofolato Redutase (NADPH2) , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Mutação Puntual/genética , Mutação Puntual/fisiologia , Reação em Cadeia da Polimerase , Espectrometria de FluorescênciaRESUMO
A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5 in the presence of a 3'-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 degrees C; if the single-base mutation is present, the probe melts at 57 degrees C. Concurrent amplification and analysis from genomic DNA takes 20-45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
