They are kept at 4☌ and protected from light.Įthanol, magnesium chloride, sodium citrate, Trizma base, sodium chloride, EDTA, formamide, hydrochloric and sulphuric (95–97%) acids are of analytical grade. Working solutions are conserved at 4☌.ģ mM 3-indoxyl phosphate (3-IP) solutions are daily prepared in a 0.1 M Tris-HCl buffer, 10 mM MgCl 2, pH 9.8. Aliquots are prepared and maintained at –20☌. Hybridisation takes place in a 2×SSC (saline sodium citrate, 30 mM sodium citrate buffer with 300 mM sodium chloride and pH 7.0) buffer containing 50% of formamide.Īlkaline-phosphatase (AP) labelled streptavidin (ST-AP) is prepared in 0.1 M Tris-HCl buffer, 1 mM MgCl 2, pH 7.2. Oligonucleotide solutions are prepared in TE buffer pH 8 (0.1 M Tris-HCl buffer solution, 1 mM in EDTA). The thiol group is separated to the first base by an aliphatic linker of three carbons (103 nmol): 5′-CTT-TTT-CTT-TTT-GTC-CTT-TTT-AGG-CTC-TGT-3′-(CH 2) 3-SH – Biotinylated 3-base mismatch target (184 nmol): 5′-ACA-G CG-CCT-AAA-AA C-GAC-AAA-AAG-A GA-AAG-3′-biotin.– Biotinylated target (193 nmol): 5′-ACA-GAG-CCT-AAA-AAG-GAC-AAA-AAG-AAA-AAG-3′-biotin.Both strands are biotinylated at the 3′ end for allowing hybridisation detection.
A three-base mismatch strand with mismatches located in bases number 5, 15 and 26. This means an improvement of various orders of magnitude when compared with limits of detection reported in the bibliography for DNA assays.Ī target sequence corresponds to a portion of SARS virus, bases exactly comprise between 2927, both included. The limit of detection, calculated as the concentration corresponding to a signal which is three times the standard deviation of the intercept, results to be 5 pM. From the results of hybridization assay and recording of the analytical signal no significant difference found between the analytical signal obtained for a 3.03nM solution of the complementary target strand and three-base mismatch strand. Genosensor construction include following steps: a drop of 5 mL of 1.02 mM thiolated probe deposited on the gold film and maintain at 37☌ for 20 min or at 41☌ for 12 h it is cleaned with 0.1M Tris-HCl buffer further a15 mL drop of a 2% 1-hexanethiol solution is deposited on the gold film and maintained for 10 min and again cleaned with a 2×SSC buffer solution pH 7. The chapter tests the sensitivity and the selectivity of the SARS genosensor using complementary strands of SARS virus and three-base mismatch strands. This chapter presents a procedure for the construction of a hybridization-based genosensor for a SARS (severe acute respiratory syndrome) virus sequence on a 100nm sputtered gold film, which works as immobilization and transduction surface.
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