Practice Questions
A student argues that since DNA is negatively charged, it should be loaded at the anode end of the gel electrophoresis apparatus. Critique this statement.
To isolate DNA from a fungal cell, which specific enzyme would you apply to break down its cell wall?
Apply the standard convention for naming restriction enzymes to explain the origin of the name 'EcoRI'.
Name the enzyme used to join the ends of cut DNA molecules to form recombinant DNA.
Define 'biotechnology' as given by the European Federation of Biotechnology (EFB).
Examine the following DNA sequence: . Explain why it is considered a palindromic sequence in the context of molecular biology.
Name the scientists who constructed the first artificial recombinant DNA molecule in 1972.
What is the function of a bioreactor in biotechnology?
Compare and contrast the functions of exonucleases and endonucleases in the context of DNA manipulation.
Justify the use of a thermostable DNA polymerase, like Taq polymerase, in PCR instead of a regular DNA polymerase from E. coli.
You are given a mixture of DNA fragments with sizes 500 bp, 2000 bp, and 3500 bp. Design how you would separate and visualize these fragments using gel electrophoresis. Predict the outcome and justify the principle behind the separation.
What is a palindromic sequence in DNA?
Critique the use of a single restriction enzyme for cutting both the vector and the foreign DNA. Is it always necessary? Justify your answer.
A lab performs a PCR reaction to amplify a specific DNA segment. After 30 cycles, they find no amplified product on the agarose gel. Propose three distinct potential reasons for this failure, evaluating the role of each component in the PCR process.
Formulate a hypothesis explaining why eukaryotic cells do not possess restriction endonucleases naturally.
Analyze the role of the 'ori' site in a cloning vector.
Describe the convention for naming restriction enzymes, using EcoRI as an example.
Describe the steps involved in the isolation of DNA from a bacterial cell. Mention the enzymes used to remove other macromolecules like RNA and proteins.
Define 'downstream processing'.
Describe the process of separating DNA fragments using agarose gel electrophoresis.
Describe the 'heat shock' method for making a host cell competent to take up recombinant DNA.
List two methods, other than heat shock, for introducing alien DNA into host cells.
If a single molecule of double-stranded DNA is subjected to PCR, calculate the number of DNA molecules that would be produced after 5 cycles, assuming 100% efficiency. Analyze the nature of the amplification process.
Demonstrate how the Polymerase Chain Reaction (PCR) is used to amplify a specific DNA segment. Your answer should analyze the role of primers, Taq polymerase, and the three key temperature-dependent steps.
Analyze the principle behind the separation of DNA fragments using agarose gel electrophoresis. Explain why DNA moves towards the anode and how fragments are separated based on size.
Examine the significance of downstream processing in biotechnology. List the key steps involved after the biosynthetic stage and explain why they are critical before a product can be marketed.
A scientist wants to transform E. coli cells with a recombinant plasmid. Apply your knowledge of the process to explain the steps required to make the bacterial cells competent and induce the uptake of the plasmid DNA.
Contrast traditional hybridisation techniques with modern genetic engineering (recombinant DNA technology) in terms of gene manipulation and specificity.
Explain the two core techniques that enabled the birth of modern biotechnology.
Examine the complete process of creating a recombinant DNA molecule. Starting from a source DNA and a plasmid vector, describe the key enzymes involved and their specific actions that lead to the formation of a chimeric DNA molecule.
A researcher wants to clone a gene of interest into the pBR322 vector. They have two options for restriction sites: Pst I (within the ampicillin resistance gene, ) and Sal I (within the tetracycline resistance gene, ). Propose which site is better to use and formulate a screening process to identify the recombinant colonies.
Propose a method to introduce a gene for pest resistance into a dicot plant. Justify your choice of vector and explain the natural mechanism that your proposed method exploits.
A scientist wants to express a eukaryotic gene in a prokaryotic host like E. coli. The eukaryotic gene contains introns. Evaluate the direct use of genomic DNA from the eukaryote for this purpose. Propose a more effective strategy to achieve successful expression.
Critique the method of 'insertional inactivation' for screening recombinants using antibiotic resistance genes (e.g., in pBR322). Propose an alternative method that is more efficient and justify why it is an improvement.
Formulate a comprehensive quality control plan for a newly developed recombinant protein therapeutic (like insulin) before it can be marketed. Your plan should cover stages from downstream processing to final product formulation, justifying the necessity of each step.
Evaluate the significance of 'sticky ends' versus 'blunt ends' in the process of creating recombinant DNA. Propose a scenario where using a restriction enzyme that creates blunt ends might be advantageous, and explain how the ligation process would differ.
Design an experiment to create a recombinant E. coli bacterium capable of producing human insulin. Your design must outline the key steps, the specific tools (enzymes, vector), and the method for selecting the transformed bacteria. Justify your choice of vector and selection method.
Explain the process of 'insertional inactivation' as a method for selecting recombinants.
Design a protocol for isolating pure plasmid DNA from a bacterial culture. Your protocol should detail the enzymes required to break down the bacterial cell components and the chemical treatment needed to precipitate the DNA, while ensuring minimal contamination from chromosomal DNA and RNA.
Explain the three main steps of a single cycle of the Polymerase Chain Reaction (PCR). Also, name the specific type of DNA polymerase used and explain why it is suitable for this process.
Explain the key features required to facilitate cloning into a vector. List at least three essential features and describe their functions.
A researcher ligates a foreign DNA fragment into the BamH I site of the pBR322 vector. Analyze the expected growth pattern when non-recombinant, recombinant, and non-transformed E. coli cells are plated on different antibiotic media.
Evaluate the role of bioprocess engineering in biotechnology. Compare a simple stirred-tank bioreactor with a sparged stirred-tank bioreactor. Create a scenario where one type would be critically preferred over the other for producing a specific recombinant protein.
Analyze the alternative method for selecting recombinants that uses insertional inactivation of the -galactosidase gene. Explain how recombinant and non-recombinant colonies are visually differentiated and why this method is considered less cumbersome.
Compare and contrast the features and functions of a simple stirred-tank bioreactor with a sparged stirred-tank bioreactor. Analyze the advantages these bioreactors offer over simple shake flasks for large-scale production.