Polymerase chain reaction (PCR) is one of the most powerful techniques in a variety of clinical and biological research fields. In this paper, a chemometrics approach, combining experimental design (ED) and artificial neural network (ANN), was proposed for optimization of PCR amplification of lycopene cyclase gene carRA in Blakeslea Trispora. Five-level star design was carried out to obtain experimental information and provide data source for ANN modeling. Nine variables were used as inputs in ANN, including the added amount of template, primer, dNTP, polymerase and magnesium ion, the temperature of denaturating, annealing and extension, and the number of cycles. The output variable was the efficiency (yield) of the PCR. Based on the developed model, the effects of each parameter on PCR efficiency were predicted and the most suitable operation condition for present system was determined. At last, the validation experiment was performed under the optimized condition, and the expectant results were produced. The results obtained in this paper showed that the combination of ANN and ED provided a satisfactory optimization model with good descriptive and predictive abilities, indicating that the method of combining ANN and ED can be a useful tool in PCR optimization and other biological applications.
Summary.In this article, a rapid, novel, and effective method was presented to separate pure steviol glycosides from leaves of Stevia rebaudiana Bertoni, including stevioside, rebaudioside A, and rebaudioside C, using resin-based column chromatography followed by preparative high-performance liquid chromatography under hydrophilic interaction liquid chromatography (HILIC) mode. The separation procedure was first optimized on an analytical HILIC column and then scaled up on a preparative HILIC column using a mobile phase consisting of 83% acetonitrile in water with flow rate of 10 mL⋅min −1 at 25°C. 200 mg of the crude extract containing 68.1% steviol glycosides yielded 79.2 mg stevioside, 9.4 mg rebaudioside C, and 33.7 mg rebaudioside A, with the purities of 97.5%, 96.8%, and 97.2%, respectively. Those products were identified by MS, 1 H NMR, and 13 C NMR.
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