Question 1: Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
|Human Growth Hormone||Used for treating dwarfism|
|Human Insulin||Used in case of insulin dependent diabetes.|
|Follicle Stimulating Hormone||Treatment of fertility disturbance|
|Factor VIII: Kogenate||Treatment of bleeding in haemophilia|
|Erythropoietin||Treatment of anemia; commonly associated with chronic renal failure and cancer.|
|Granulocyte colony stimulating factor||Treatment of low neutrophil count in blood|
|Alpha-galactosidase||Treatment of Fabry Disease|
|Tissue plasminogen activator||Treatment of blood clot|
|Glucocerebrosidase||Treatment of Gaucher’s disease|
|Interferon||Treatment of multiple sclerosis|
Question 2: Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.
Question 3: From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
Answer: We know that enzyme is a protein, and protein is synthesized from a small strand of DNA. Hence, DNA is bigger than enzymes in terms of molecular size.
Question 4: What would be the molar concentration of human DNA in a human cell? Consult your teacher.
Answer: Molar concentration of human DNA in a human cell (diploid)
= Number of chromosome × 6.023 × 1023
= 46 × 6.023 × 1023
= 2.77 × 1021 mole
Question 5: Do eukaryotic cells have restriction endonucleases? Justify your answer.
Answer: The DNA of eukaryotes is highly methylated. Methylation protects the DNA from the activity of restriction enzymes. Hence, eukaryotic cells do not have restriction endonucleases.
Question 6: Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Answer: Stirred tank bioreactors have following advantages over sake flasks:
- Facility of taking out samples in small volume.
- Foam beaker regulates the amount of foam.
- Temperature and pH can be controlled.
Question 7: Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Answer: Following are 5 examples of palindromic DNA sequences:
5’ – AGCT – 3’ (3’ – TCGA – 5’)
5’ – GTTAAC – 3’ (3’ – CAATTG – 5’)
5’ – TTCGAA – 3’ (3’ – AAGCTT – 3’)
5’ – CAGCTG – 3’ (3’ – GTCGAC – 5’)
5’ – GACGTC – 3’ (3’ – CTGCAG – 5’)
Question 8: Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Answer: Recombinant DNA is made during Pachytene stage of Prophase I of Meiosis I. This is the stage when crossing over of chromosomes takes place; which involves exchange of segments between non-sister chromatids.
Question 9: Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?
Answer: Reporter gene act as a selectable marker to determine whether the host cell has taken up the foreign DNA or not. For this, reporter gene and foreign gene are placed in the same DNA construct.
Question 10: Describe briefly the followings:
(a) Origin of replication
Answer: The sequence from where replication starts in the DNA is called the Origin of Replication (ori). When a piece of DNA is linked to this sequence, it can be made to replicate within the host cell.
Answer: For producing large volumes of culture, bioreactors are used. Stirring type bioreactors are the most commonly used bioreactors. It is generally cylindrical with a curved base. The stirrer facilitates even mixing and oxygen availability through the reactor. A good quality bioreactor has an agitator system, a temperature control system, pH control system and sampling ports.
(c) Downstream processing
Answer: Downstream processing involves separation and purification of the finished product. If the finished product is a drug, it has to undergo thorough clinical trials. Strict quality control measures are also required.
Question 11: Explain briefly
Answer: PCR stands for Polymerase Chain Reaction. Multiple copies of the gene of interest are synthesized in vitro during this reaction. It is achieved by using two sets of primers and the enzyme DNA polymerase. (Small chemically synthesized oligonucleotides which are complementary to the regions of DNA are called primers).
(b) Restriction enzymes and DNA
Answer: The enzymes which cut the DNA are called restriction enzymes. Hind II was the first restriction endonuclease to be discovered. Hind II always cuts DNA molecules at a particular point by recognizing a specific sequence of six base pairs. This specific base sequence is called the recognition sequence for Hind II. Today, more than 900 restriction enzymes are known. Different enzymes recognize different sequences.
Answer: Chitinase is an enzyme which facilitates degradation of chitin. This enzyme is used while extracting DNA from a fungus.
Question 12: Discuss with your teacher and find out how to distinguish between
(a) Plasmid DNA and Chromosomal DNA
|Plasmid DNA||Chromosomal DNA|
|It is extra-chromosomal DNA in bacteria.||Chromosomal DNA represents the entire DNA of an organism, present inside the chromosome.|
(b) RNA and DNA
|Has single strand.||Has double strand.|
|Contains ribose sugar.||Contains deoxyribose sugar.|
|Cannot replicate.||Can replicate itself.|
|Is a component of ribosome.||Is a component of chromosome.|
(c) Exonuclease and Endonuclease
Answer: Exonuclease removes nucleotides from the ends of the DNA, while endonuclease: makes cuts at specific positions within the DNA.