Published in Scientific Bulletin. Series F. Biotechnologies, Vol. XXV, Issue 1
Written by Valentina VASILE, Matilda CIUCĂ, Elena NICOLAE, Cătălina VOAIDEȘ, Călina Petruța CORNEA
Although it is well known that molecular biology techniques have recently seen a significant increase in the development of new research methods, an important role when choosing the appropriate working methods lies not only in the methods acceptance criteria such as specificity, applicability, robustness but also in the final cost of the implemented technology. Therefore, although methods involving SSR markers are much more specific and informative than other and these markers can be easily identified by PCR techniques, difficulties may arise in interpreting the results if the chosen SSR markers are not suitable for researches purposes or the conditions for PCR reaction are not appropriate. The methods optimization plays an important role in obtaining the desired results. The aim of this study was to choose the most suitable PCR conditions optimizing either the reaction parameters by varying the reagents used or their concentration as well as PCR amplification conditions when using fourteen SSRs markers in wheat cultivars grown in Romania. The SSRs markers DuPw167, DuPw217, DuPw004, DuPw115, DuPw205, Xgwm155, Xgwm413, Xgwm003, Xgwm372, Xbarc184, Xbarc347, Xbarc074, Xgwm052 and Xgwm095 were amplified with three PCR reaction mixtures at different annealing temperatures. All SSRs markers gave the best amplification profiles with the PCR mix 2 which had as reagent a Hot Start Taq DNA Polymerase. By choosing the best methods conditions a successful amplification and a unique allele profile may be provided thus being able to standardize the methods used.
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