Single tube optimisation of APOE genotyping based on melting curve analysis Francesc Francés a,b, ⁎ , Olga Portolés c , José Vicente Sorlí a,b , Marisa Guillén a , José I González a,b , Dolores Corella a,b a Genetic and Molecular Epidemiology Unit. Department of Preventive Medicine. School of Medicine. University of Valencia, Valencia, Spain b CIBER Fisiopatología de la obesidad y Nutrición. Instituto de Salud Carlos III, Spain c Department of Information Systems and Languages. Jaume I University, València, Spain Received 29 January 2008; received in revised form 12 March 2008; accepted 12 March 2008 Available online 8 April 2008 Abstract Objectives: To develop and validate a single-tube protocol for APOE genotyping using fluorescent probes. Design and methods: We have designed and validated a hybrid, single-tube, SimpleProbe/FRET probe protocol that takes advantage of the different probe wavelength emissions. Results: Our method offers high quality outcomes, minimum interferences between probe signals and is 100% concordant with the reference protocol. Conclusions: This method is cheaper, faster and more reliable and versatile than other alternatives proposed. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Genotype; Analysis; Validation studies APOE is one of the most well-known apolipoproteins linked to lipid profile and cardiovascular disease. There are three common codominant alleles called ε2, ε3 and ε4 [1]. These alleles arise from base substitutions in codons 158 and 112 [2]. Classically, APOE genotyping has been undertaken using allele-specific oli- gonucleotide hybridization [3], oligonucleotide ligation assay [4], single strand conformational polymorphism analysis [5], ampli- fication refractory mutation system [6], and the reference RFLP- PCR method [7]. In recent years, further techniques have been developed. One of the most successful alternatives is the APOE genotyping using FRET (Fluorescent Resonance Energy Trans- fer) probes with the LightCylcer device, based on melting curve analysis (MCA) [8,9]. The FRET system consists of an oligo- nucleotide complementary to the wild or the mutated sequence, containing the polymorphic base in the middle of its sequence, and has the LC-red640 as the fluorophore at its 5′ end. A second hybridization primer (anchor primer) is located upstream at a distance of one to three nucleotides, and is labeled with fluo- rescein at its 3′ end. MCA genotyping is based on the different melting tempera- tures of the same probe when different alleles are present. Recently, high-throughput genotyping using fluorescence MCA is commercially available as LightTyper™ (Roche, Mann- heim, Germany). The instrument [10] offers a higher throughput method than the original LightCycler™. The LightTyper is ex- plicitly designed for melting curve analysis to perform a fast, straight-forward, and reliable allelic discrimination. The sys- tem provides post-amplification genotyping in 10–15 min, using 384-well sample plates and performs genotyping automatically. A variety of probe chemistries are compatible for genotyping, including single-labeled probes, and FRET probes. SimpleP- robes™ are designed to specifically hybridize into a target se- quence that contains the polymorphism of interest [11]. In both types of probes, their melting from the target sequence produces a clear reduction in the fluorescence emitted by the probe. SimpleProbes are more cost-effective than FRET probes and Available online at www.sciencedirect.com Clinical Biochemistry 41 (2008) 923 – 926 ⁎ Corresponding author. Department of Preventive Medicine. School of Medicine, Avda. Blasco Ibañez, 15, 46010-Valencia, Spain. Fax: +34 963864166. E-mail address: francesc.frances@uv.es (F. Francés). 0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.03.010