Development of multiplex PCR system for identification of glyphosate-tolerant sugar beet
DOI:
https://doi.org/10.21498/2518-1017.4(33).2016.88686Keywords:
gene cp 4 epsps, 35S promoter, the transgenic sugar beet, amplification parametersAbstract
Purpose. To create a multiplex system for identification glyphosate-tolerant sugar beet by using PCR.
Methods. Molecular genetic analysis.
Results. The article presents the results of studies to determine the parameters of the polymerase chain reaction (PCR) in order to develop a multiplex system for identification of the structural elements of the design of transgenic gene cp 4 epsps, which provides tolerance to glyphosate. For amplicon target DNA sequences, the following values of temperature conditions of PCR were determined: step 1 (initial denaturation) 95 °C – 3 min; step 2 (specific reaction products accumulation): denaturation 95 °C – 45 s; hybridization of primers 55 °C – 50 s; elongation 72 °C – 1 min; number of cycles – 40; step 3 (final elongation) 72 °C – 6 min. A series of PCR were carried out for the purpose of selecting the optimal amount of DNA matrix for efficient estimate of transgenic sugar beet plants for the presence of specific sequences.
Conclusions. To identify transgenic glyphosate-tolerant sugar beet, it is advisable to determine 35S promoter and gene cp 4 epsps in individual genotypes. It was found that during the selection of temperature parameters of multiplex reaction a 5 °C rise in primer hybridization temperature did not affect the identification of gene als that allowed to include specific primers for determination of this sequence as an internal control. Based on the results of test multiplex reactions, concentrations of dNTPs and Mg2+ ions were determined that allowed to exclude the possibility of non-specific fragments and false-negative results. The optimum amount of matrix DNA (100–150 ng) for an efficient estimate of transgenic sugar beet plants for the presence of specific sequences was determined. Obtained results allowed to develop a multiplex test system for identification of transgenic glyphosate-tolerant sugar beet which can be used for simultaneous determination of the 35S promoter, cp 4 epsps gene and als gene as an internal reaction control.
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Ukrainian Institute for Plant Varieties Examination 
Селекційно-генетичний інститут
Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine
The National Academy of Agrarian Sciences of Ukraine