Primer was synthesized with 5´ C6 amino linker by The Midland Certified reagent Company Inc. (Midland, TX, USA). Synthesis was validated using each pair of oligonucleotides by RT-PCR and found to yield PCR products of the expected size.
Label
6 FAM
Label protocol
Peformed during the synthesis of the oligonucleotide by The Midland Certified reagent Company Inc. (Midland, TX, USA).
Hybridization protocol
DNA microarray chip was prepare for hybridization by washing extensively with 0.1% SDS, rinsed twice with double deionizer water and heated in double deionizer water for 3 min at 95° C. After blocking with prehybridization buffer (4X SSC, 1 %SDS nd 10 mg/ml BSA) for 45 min at 42° C, DNA microarray chip was washed with double deionizer water for 20 s and dried under filtered air flow. A volume of 5 µl containing specific primer for serotype was used. The microarray substrates and coverslips were sealed in a chamber from BIO-RAD (Bio-Rad Laboratories, Inc., Ca., USA) and incubated at 42o C for 16-18 h at high humidity. Coverslip and supports were gently removed (4× SSC in a wash bottle) and arrays were washed by immersion into 1× SSC, 0.1% SDS for 10 min (10 slides in 250 ml), 0.1× SSC, 0.1% SDS twice for 10 min and 0.1× SSC twice for 10 min. Finally, blow the slide dry with air by pushing the liquid away from the spots and towards the outer edges.
Scan protocol
Slides were scanned using Scanner GeneTac LS IV (Genomic Solutions, Molecular Dynamics Generation III) to detect FAM, fluorescence. For the detection of DENV serotypes we used the excitation lines at 494 nm wavelength of a laser scanner (Scanner Gene TAC LSIV from Genomic Solutions) by adding pixel intensities of each spot image
Data processing
Data was normalized by experiment mean after background correction.
