title: Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.
abstract: 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/μL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/μL), 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.
authors.affiliation: Department of Hematology, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Martinez-Serra
authors.affiliation: Department of Clinical Analysis, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Robles
authors.affiliation: ECOGEN, Barcelona, Spain.
authors.forename: Antoni
authors.initials: A
authors.lastname: Nicolàs
authors.affiliation: Department of Hematology, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Gutierrez
authors.affiliation: Department of Hematology, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Ros
authors.affiliation: Department of Hematology, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Amat
authors.affiliation: Department of Cell Biology, University of the Balearic Islands, Palma de Mallorca, Spain.
authors.forename: Regina
authors.initials: R
authors.lastname: Alemany
authors.affiliation: Department of Cell Biology, University of the Balearic Islands, Palma de Mallorca, Spain.
authors.forename: Oliver
authors.initials: O
authors.lastname: Vögler
authors.affiliation: Department of Clinical Analysis, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Aina
authors.initials: A
authors.lastname: Abelló
authors.affiliation: Department of Clinical Analysis, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Aina
authors.initials: A
authors.lastname: Noguera
authors.affiliation: Department of Hematology, Hospital Universitary Son Espases, Palma de Mallorca, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Besalduch
keywords: FRET; LightCycler® 2.0 Instrument; erythrocytes; lysis; melting peak; real-time PCR; white blood cells