Detection of the cp4 epsps Gene in Maize Line NK603 and Comparison of Related Protein Structures: An Advanced Undergraduate Experiment

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• 作者：Nicole K. Swope ; Patrick J. Fryfogle ; Tami L. Sivy
• 刊名：Journal of Chemical Education
• 出版年：2015
• 出版时间：July 14, 2015
• 年：2015
• 卷：92
• 期：7
• 页码：1229-1232
• 全文大小：214K
• ISSN：1938-1328

文摘

A flexible, rigorous laboratory experiment for upper-level biochemistry undergraduates is described that focuses on the Roundup Ready maize line. The work is appropriate for undergraduate laboratory courses that integrate biochemistry, molecular biology, or bioinformatics. In this experiment, DNA is extracted and purified from maize kernel and leaf samples collected from a Roundup Ready maize grower鈥檚 field. A small segment of DNA (108 base pairs) specific to the Roundup Ready transgene that codes for CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) is amplified with polymerase chain reaction (PCR) to detect the presence of the gene in the maize samples. Students additionally choose a protein closely related to CP4 EPSPS as determined by amino acid sequence alignments. The selected amino acid sequences are submitted to an online protein modeling program where students compare their protein with the herbicide-resistant enzyme found in Roundup Ready crops. This experimental paradigm gives students a physical appreciation for the central dogma of biology, as they are exposed to products derived from the replication, transcription, and translation events belonging to a genetically modified crop. The PCR portion of the laboratory allows students to perform an in vitro replication of a portion of the NK603 transgene and identify the segment via ultraviolet radiation. In direct connection to the PCR portion, the protein structure elucidation gives rise to the central ideas of evolution鈥攖hat slight changes in the genetic code of DNA, translated into proteins, produce novel protein structures with significantly different function.