Syringe

Biotechnology: Principles and Processes

EXERCISE SOLUTIONS

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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).

Ans:

Human insulin – Diabetes

DNase I – Treatment of cystic fibrosis.

Chronic Gonadotropin – Treatment of infertility

Human growth hormone – Dwarfism cure

Calcitonin – Treatment of rickets

TPA (tissue plasminogen activator) – Heart attack/strokes

PDGF (platelet derived growth factor) – Stimulates wound healing.

Interferon (α, β and γ) – Treatment of viral infection and cancer.

Interlinkins – Enhances activity of  immune reaction,

Reo Pro – Prevention of blood clots.

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.

Ans:

Name of the Restriction enzyme – Bam HI.

The substrate DNA on which it acts –

rDNA.jpg

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?

Ans: Even though both DNA and enzymes are macromolecules, DNA is bigger in molecular size as compared to proteins. Because DNA is made up of sugar, phosphate and nitrogenous bases. An enzyme is made up of only proteins, so no complexity of molecules.

 

Question 4. What would be the molar concentration of human DNA in a human cell? Consult your teacher.

Ans: The molar concentration of DNA in human cell is 2.77 x 1023 moles of cell extract.

 

Question 5. Do eukaryotic cells have restriction endonucleases? Justify your answer.

Ans: Eukaryotic cells have no restriction enzymes as the DNA molecules of eukaryotes are heavily methylated. All the restriction endonucleases have been isolated from various strain of bacteria.

Question 6. Besides better aeration and mixing properties, what other advantages do stirred-tank bioreactors have over shake flasks?

Ans: Shake flasks are used for growing and mixing the desired materials on a small scale in the laboratory. A large-scale production of desired biotechnological product is done by using ‘bioreactors’. Besides better aeration and mixing properties, the bioreactors have following advantages;

  • Stirred-tank bioreactor facilitate temperature control system, pH control system, foam control system.

  • It has sampling ports to obtain small volumes of cultures periodically from die reactor for sampling.

  • Facilitates even mixing and oxygen availability throughout the bioreactor.

 

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.

Palindrome nucleotide sequences in the DNA molecule are groups of bases that form the same sequence when read both forward and backward. Five examples of palindromic DNA sequences are as follows:

(i) 5′——GGATCC——3’

     3′——CCTAGG——5′

(ii) 5’——AAGCTT——3′

      3′——TTCGAA ——5′

 

(iii) 5′——ACGCGT——3′

        3′——TGCGGA—— 5′

(iv) 5′——ACTAGT——3′

       3′——TGATCA——5′

(v) 5′——AGGCCT——3′

      3′——TCCGGA——5′

Question 8. Can you recall meiosis and indicate at what stage a recombinant DNA is made?

Ans: Recombinant DNA occurs during pachytene stage of prophase of meiosis I by between non-sister chromatids of homologous chromosome.

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?

Ans: A reporter enzyme can be used to differentiate transformed cells by tracking down the activity of its co-responding genes (receptor gene). For e.g., (3-galactosidase (Lac Z) activity is not found in transformed cells so that they appear white in colour. The others, which appear blue in colour, indicate that cells do not carry foreign DNA.

 

Question 10. Describe briefly the followings:

a. Origin of replication

b. Bioreactors

c. Downstream processing

 

a. Ori: This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.

b. Bioreactors: Bioreactors are vessels of large volumes (100-1000 litres) in which raw materials are biologically converted into specific products.

It provides all the optimal conditions for achieving the desired product by providing optimal growth conditions like temperature, pH, substrate, salt, vitamins and oxygen.

Bioreactor has the following components: An agitator system, An oxygen delivery system, Foam control system, Temperature control system, pH control system, Sampling ports to withdraw cultures periodically.

c. Downstream processing: The product obtained is subjected to a series, of processes collectively called downstream processing before it is made into a finished product ready for marketing. The two main processes are separation and purification. The product is then formulated with suitable preservatives. Such formulations have to undergo clinical trials, in case of drugs.

Question 11. Explain briefly

a. PCR

b. Restriction enzymes and DNA

c. Chitinase

a. PCR: It stands for polymerase chain reaction, it’s a method of amplification of small segments of DNA.

PCR involves 3 steps:

(i) Denaturation of desired double strand DNA-to ssDNA.

(ii) Annealing of primer to ssDNA (single standard).

(iii) Extension of primer by Taq DNA polymerase isolated form Thermus aquaticus.

b. Restriction enzymes and DNA: Restriction enzymes is a group of enzymes used to cleave or cut DNA strands each having a characteristics base sequence at which it cleaves.

Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds; exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.

 

c. Chitinase: During the isolation of DNA in the processes of recombinant DNA technology, the fungal cell is heated with an enzyme called chitinase. The chitinase enzyme dissolves the chitin membrane to open the cell for release of DNA along with other macromolecules such as RNA, proteins, polysaccharides and lipids.

 

Question 12. Discuss with your teacher and find out how to distinguish between

a. Plasmid DNA and Chromosomal DNA

b. RNA and DNA

c. Exonuclease and Endonuclease

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Important Questions

 
  1. a. Differentiate Endonucleases and Exonucleases. (2 marks)
    b. Diagrammatically represent recombinant D.N.A technology. (3 marks)
  2. Name the plasmid present in Agrobacterium tumefaciens. (1 mark)
  3. With an example, explain the convention for naming restriction endonucleases scientifically. (2 marks)
  4. a. Mention four tools required for Recombinant DNA technology. (2 marks)
    b. With reference to gel electrophoresis, what is elution? (1 mark)
  5. Write the restriction site for EcoRI enzyme. (1 mark)
  6. Write the methods to introduce alien DNA into host cells. (2 marks)
  7. Explain the structure of pBR322 with a neat labelled diagram. (5 marks)
  8. Draw a labelled diagram of simple stirred tank bioreactor. (5 marks)
  9. Write the function of DNA Ligase. (1 mark)
  10. Name the technique involved in separation and isolation of DNA fragment. Which dye is used to stain to make the DNA visible under UV light. (2 marks)
  11. What is plasmid? Mention two sites of plasmid. (3 marks)
  12. Define biotechnology. (1 mark)
  13. Explain the steps involved in rDNA technology. (5 marks)
  14. What is polymerase chain reaction? Name the bacterium from which the polymerase enzyme
    used in this technique is obtained. Write the schematic representation of this technique. (5 marks)
  15. Mention any three features of vectors that are most suitable for the purpose for recombinant
    DNA technology. (3 marks)
  16. Mention two classes of nucleases. Suggest their respective roles. (2 marks)
  17.  What is the uniqueness of Taq polymerase? (1 mark)
  18.  Explain how DNA is isolated from cells. (3 marks)