Determining optimal amplification parameters and assessing specificity of developed primers

Abstract

Objective: The aim of the study is to determine optimal amplification parameters and assess specificity of developed primers. Methods: Algorithmic analysis of sequences has been performed, and optimal locations for primers have been searched for the use of the PrimerQuest application with indication of the maximum size of the amplicon read by a couple of primers in the settings. Among the variants of primers proposed by the application, optimal pairs have been chosen for each type of fruit-and-berry raw materials, with regard to such parameters as length of the primer, annealing temperature, and amplicon location. Results: The optimal parameters of the amplification process have been defined volume of primers and amplification mode. The chosen mode of amplification was confirmed by the results of the polymerase chain reactions with all the samples of the fruit-and-berry raw materials with visualization in the form of the electrophoretogram. Additional check of primers specificity has been performed with the use of the BLAST algorithm. Conclusion: It has been determined that all sequened fragments that can be read by each of the pairs of the developed primers coincided with those deposited in the GeneBank by sequences of the studied raw materials.