The first recombinant DNA was constructed by linking an antibiotic resistance gene with a native plasmid of:
In EcoRI, the letter R stands for:
DNA fragments in agarose gel electrophoresis move towards the:
Separated DNA bands are visualised by staining with ethidium bromide and exposing to:
Taq polymerase is obtained from:
In pBR322, the gene for ampicillin resistance contains the restriction sites:
Biolistics uses microparticles of:
The Ti plasmid used as a vector for plants comes from:
Bacterial cells are made competent to take up DNA by treatment with:
In blue-white screening, colonies with recombinant plasmids appear:
What are sticky ends? Why are they useful in genetic engineering? (2 marks)
Why is the origin of replication essential in a cloning vector? (2 marks)
What is elution? Why is it done after gel electrophoresis? (2 marks)
Why are restriction enzymes called molecular scissors and DNA ligase called molecular glue? (2 marks)
Why is a single recognition site preferred for a restriction enzyme in a vector? (2 marks)
How do retroviruses become useful vectors after being disarmed? (2 marks)
Why do the cut ends of the vector and the foreign DNA need to be produced by the same restriction enzyme? (2 marks)
Distinguish between exonucleases and endonucleases. (2 marks)
Why is a continuous culture system better than a batch culture for large-scale production? (2 marks)
What is a selectable marker? Name two commonly used markers for E. coli. (2 marks)
Calculate the expected frequency of occurrence of the EcoRI site (GAATTC) in a random DNA sequence. How many sites would be expected in the E. coli genome of about 4.6 million bp? (Take 4^6 = 4,096.) (3 marks)
A PCR is run for 30 cycles starting with 10 template molecules. Calculate the theoretical number of copies (take 2^30 = 1.07 x 10^9). If each cycle takes 4 minutes, how long will the reaction take? (3 marks)
A linear DNA of 10 kb is digested with an enzyme that cuts at 2 kb, 5 kb and 8 kb from one end. List the fragment sizes and state the order in which they appear on a gel from the well downwards. (3 marks)
A bioreactor of 1,000 litres is filled to 80% of its volume with medium. If each litre yields 0.5 g of a recombinant protein, of which 70% is recovered after downstream processing, calculate the final mass of protein. (3 marks)
A plasmid vector of 3,000 bp has one EcoRI site and one BamHI site 600 bp apart. A gene is inserted after cutting with both enzymes. If the gene is 1,500 bp, calculate the size of the recombinant plasmid. (3 marks)
Read the passage and answer the questions. A researcher wants to clone a human gene into E. coli. She cuts the gene and the plasmid with EcoRI, mixes them with DNA ligase, and transfers the mixture into bacteria treated with ice-cold calcium chloride followed by a brief incubation at 42 degrees C. (i) Why did she use the same enzyme for both DNAs? (ii) What is the purpose of the heat shock? (iii) How will she identify bacteria carrying the recombinant plasmid? (5 marks)
Read the passage and answer the questions. A forensic lab receives a tiny blood sample from a crime scene, containing very little DNA. The analyst amplifies a target region using two primers, deoxynucleotides and a thermostable enzyme over 30 cycles. (i) Name the technique. (ii) Describe the three steps of each cycle. (iii) Why must the enzyme be thermostable? (5 marks)
Read the passage and answer the questions. After digesting DNA with a restriction enzyme, a student loaded the sample in wells of an agarose gel and applied an electric field. After staining, she saw bright orange bands at different distances from the wells. (i) Why do DNA fragments move through the gel? (ii) Why do fragments of different sizes move different distances? (iii) What gives the orange colour to the bands? (5 marks)
Read the passage and answer the questions. A company producing a recombinant enzyme scaled up from a 1 litre flask to a 1,000 litre stirred-tank bioreactor. It found that maintaining oxygen and temperature became critical at the larger scale. (i) Why are bioreactors needed for large-scale production? (ii) Name four parameters controlled in a bioreactor. (iii) How does a sparged stirred-tank bioreactor improve oxygen availability? (5 marks)
Read the passage and answer the questions. A plant biotechnologist wants to introduce a gene into rice cells. Agrobacterium works poorly with rice, so she uses a gene gun that fires coated microparticles into the cells. (i) Name this method of gene transfer. (ii) What are the microparticles coated with and made of? (iii) Name another method used for animal cells. (5 marks)
Describe the structure of pBR322 with a labelled diagram, and explain how insertional inactivation of the tetracycline resistance gene helps in selecting recombinants. (6 marks)
Explain the steps of recombinant DNA technology from isolation of DNA to obtaining the foreign gene product. Draw a flowchart summarising the process. (6 marks)
Explain the polymerase chain reaction with a labelled diagram showing denaturation, annealing and extension, and give three applications of PCR. (6 marks)
Describe agarose gel electrophoresis with a labelled diagram of the apparatus and a typical gel showing DNA bands. Explain how the separated DNA is recovered. (6 marks)
Draw a labelled diagram of a simple stirred-tank bioreactor and a sparged stirred-tank bioreactor. Explain their working and describe the steps of downstream processing. (6 marks)
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