By Corinne A. Michels
Molecular Genetic research is a complicated textbook to coach the speculation and perform of molecular genetic research to senior undergraduates and graduates learning genetics, molecular biology and phone biology.
This e-book makes use of a case research method, with the yeast Saccharomyces because the version genetic organism, to provide an explanation for the speculation and perform of molecular genetic research. It presents adequate info so readers might be in a position to follow the method of their very own learn undertaking.
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Additional info for Genetic techniques for biological research : a case study approach
The most common type of epitope used by molecular biologists is the peptide epitope. This is because one can easily attach such an epitope to any protein of interest using recombinant DNA technology. This is referred to as epitope tagging. For this one simply needs to construct an in-frame fusion of the ORF of the gene encoding the protein of interest toan oligonucleotide encoding the epitope sequence. Most often, the epitope is placed at the N-terminal or C-terminal end of the ORF. It is essential to test the epitope-tagged allele for function to ensure that the presence of the epitope does not interfere with the functional activity, subcellular localization, or stability of the protein.
MalB protein is an E. coli product involved in maltose transport. Itis localized to the periplasmic space and is used as a maltose carrier. Its high-affinity binding to maltose and amyloseis the basis of this purification method. Vectors for these constructions are commercially available. TheMalB portion will provide specific binding to a column-bound amylose and the MalB fusion protein will be retained by the column. The fusion protein is released from the amylose column by excess maltose. The purified fusion protein is then cleaved at the junction sitewith a site-specific protease and the protein of interest further purified by passage over an amylose column to remove the free MalB protein and any uncleaved residue.
The samples can be observed by EM to determine the presence of contaminating components. M. M. M. 1 Equilibrium density gradient analysis. Shown are the results of an equilibrium density gradient analysis carried out to determine the subcellular localization of Gaplp, the general amino acid permease, under different culture conditions (glutamate or glutamate transferred to urea). ) The density gradient is 2OY0-6O% sucrose with and without Mg2+. The upperrow of panels shows, for each fraction, the sucrose concentration (open circles) and the relative levels of Gaplp-HA (hemagglutinin-tagged Gap1 protein) as determined by Western analysis.