THE PROBLEM: You are a scientist in the research and development laboratory of a biotechnology company.You and your colleagues (you may work with up to two additional students on this worksheet) have been tasked with the following biotechnology problem:
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
THE PROBLEM: You are a scientist in the research and development laboratory of a
- Clone a human gene and mass produce the gene product in a bacterial host (Escherichia coli).
Using your textbook as a
1. You have identified the specific gene that produces the protein product that you wish to mass produce. What is the first step you need to do in order to obtain a version of this human gene that be cloned into a bacterial cell? What special enzyme, obtained from a particular kind of virus, will you need to complete this first step? (Hint: Are there differences in genes between eukaryotes and prokaryotes?)
2. Now that you have your gene available in the correct form, diagram the steps necessary to place the gene’s DNA into a plasmid vector and list all the types of enzymes needed to do this:
3. Once you have created your recombinant plasmid, what process do you use to get the plasmid into your E. coli host cell? (Hint: You have naked DNA in a test tube.)
4. We need to screen our cells to make sure they have the correct plasmid in them. If we cloned using a plasmid containing the lacZ gene at the cloning site, we can use blue/white screening. What color would the cells be that contain a recombinant plasmid?
5. Once you have identified the correct clone containing the gene you wanted, how might you increase the production of the protein product? (Hint: The β-galactosidase gene on your plasmid came from the lactose operon.)
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