Credit hours
In-class work per week |
Practice per week |
Credits |
Duration |
Total |
4 |
5 |
15 |
15 weeks |
225 hours |
Instructor
Antonio Vargas de Oliveira Figueira
Objective
To present basic methods of recombinant DNA, including nucleic acid (DNA and RNA) extraction, quantification and analysis, genetic manipulation of bacteria (Escherichia coli and Agrobacterium tumefaciens) and plants, and the analysis of gene expression. The course will present the application of these basic methods to answer in various relevant biological questions, their adoption in basic studies of Plant Biology, Physyiology and Biochemistry, and potential aplications in agriculture. The course is introductory to the methods, based on student exposure to laboratory practices, with demonstrations and small experiments, involving brief theorethical introduction to the experiments and methods, and discussion about the potential use and applications in agrienvironmental problems.
Content
Theory:
a. Microbiological manipulations of Escherichia coli e Agrobacterium tumefaciens;
b. Isolation, purification, quantification of nucleic acids (DNA and RNA);
c. Methods of nucleic acid manipulation (digestion, ligation, transformation, hybridization and cloning);
d. Fundaments of PCR;
d. Gene expression analysis (RT-qPCR);
f. Tools for sequence analysis and primers design;
g. Plant transformation by A. tumefaciens;
h. Gene expression in E. coli.
Laboratory:
1. Manipulations of Escherichia coli e Agrobacterium tumefaciens;
2. Isolation, purification, quantification of nucleic acids (DNA and RNA);
3. PCR;
4. RNA analysis and RT-qPCR;
5. Enzymatic manipulation of DNA;
6. Plant transformation by Agrobacterium tumefaciens;
7. Heterologous expression in E.coli;
8. Sequence analysis and primer design.
Bibliography
1. BRASILEIRO, A.C.M.; CARNEIRO, V. T. de C. Manual de transformação de genética de plantas. 2a. ed. Brasília, DF: Embrapa, 2015.
2. BROWN, T.A. Genomes 4. 4th ed. Garland Science, Taylor and Francis Group, New York, NY, EUA, 2019.
3. CHRISPEELS M.J.; SADAVA D.E. Plants, Genes, and Biotechnology. Sudbury: Jones and Bartlett Publishers. 2003.
4. MENCK, C.F.M.; VAN SLUYS, M.-A. Genética Molecular Básica. Ed. Guanabara Koogan. Rio de Janeiro, RJ. Brasil. 2017.
5. SAMBROOK, J.; RUSSEL, D.W. The condensed protocols: from Molecular Cloning: a Laboratory Manual. Cold Spring
Harbor Laboratory Pres, Cold Spring Harbor, NY, EUA. 2006.
6. WALKER, J.M.; RAPLEY, R. Molecular Biomethods Handbook. Human Press, Totowa, NJ, EUA, 2008.