Journal of Food, Agriculture and Environment

Modeling and maximizing AFLP pre-amplification yield using response surface methodology with covariate

Author(s):

Muhanad Walid Akash

Recieved Date: 2011-09-08, Accepted Date: 2011-11-22

Abstract:

This study was performed to model annealing temperature and [Mg⁺²] changes during AFLP pre-amplification step in order to maximize its DNA yield. Thirteen trial experiments that consist of four factorial points (53.5°C and 0.625 mM,  53.5°C and 2.625 mM, 58.5°C and 0.625 mM,  58.5°C and 2.625 mM, annealing temperature and [Mg⁺²], respectively), four axial points (56°C and 0.211 mM,  56°C and 3.039 mM, 52.5°C and 1.625 mM, 59.5°C and 1.625 mM, annealing temperature and [Mg⁺²], respectively), and five repeated center point (56 and 1.625 mM, annealing temperature and [Mg⁺²], respectively) were performed. Response surface methodology (RSM) with covariate was employed for DNA samples obtained from barley, crocus, and faba bean plants. Applying a second order regression model showed that annealing temperature and [Mg⁺²] had a significant influence on band concentration obtained from the pre-amplification process. The canonical analysis results showed that the highest band concentration was obtained with annealing temperature equal to 56.2°C and [Mg+2] equal to 1.76 mM. The contour plot of the predicted response surface confirmed this conclusion. The predicted value at the stationary point for this optimized combination was 15.22 ng/µl compared to (15.17 ng/µl) obtained from center points. This improvement in the pre-amplification step will increase the number of selective amplifications that can be performed. 

Keywords:

Annealing temperature, amplified fragment length polymorphism (AFLP) pre-Amplification, barley, central composite design (CCD), covariate, crocus, faba bean, Mg²⁺, maximization, modeling, response surface methodology (RSM)

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Journal: Journal of Food, Agriculture and Environment
Year: 2011
Volume: 9
Issue: 3&4
Category: Environment
Pages: 1144-1147

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