Por favor, use este identificador para citar o enlazar este Item:http://hdl.handle.net/20.500.12105/19956
Título
Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.
Autor(es)
Fecha de publicación
2014
Cita
Martinez-Serra J, Robles J, Nicolas A, Gutierrez A, Ros T, Amat JC, et al. Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.. J Blood Med. 2014;5:99-106.
Idioma
Inglés
Tipo de documento
research article
Resumen
Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler() 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5*10(6) WBC/mL) and purified DNA (20 ng/muL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26ᄆ0.566 versus purified DNA 24.79ᄆ0.56. Thus, when PCR was performed from WBC (5*10(6) WBC/mL) instead of DNA (20 ng/muL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.
Palabras clave
FRET | LightCycler 2.0 Instrument | Erythrocytes | Lysis | melting peak | Real-time PCR | White blood cells
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