Explain briefly
(a)PCR
(b)Restriction enzymes and DNA
(c)Chitinase
(a) PCR = Polymerase chain reaction (in vitro method) is a molecular biological technique for enzymatically replicating DNA without using a living organism, such as E. coli or yeast.
3 steps in PCR are –
(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 Thermits aquaticus.
Uses – Amplification of desired gene/gene cloning.
Advantage- More output, greater efficiency, less error prone, less human interference and cyclic and automated.
(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.
(i)It restricts foreign DNA from entering normal cell by digesting it at various recognition site. Recognition site is palindromic.
(ii)They are endonuclease and exonuclease both types.
(iii)They produces sticky ends. Cleavage site and recognition site are different from each other. Restriction enzymes therefore are believed to be a mechanism evolved by bacteria to resist viral attack and to help in the removal of viral sequences.
(c) Chitinase – Chitinase is a enzyme to digest or breakdown glycosidic bonds in chitin cell wall of fungal cell to facilitate its transformation.
Restriction enzymes that are used in the construction of recombinant DNA are endonucleases which cut* the DNA at specific-recognition sequence'. What would be the disadvantage if they do not cut the DNA at specific- recognition sequence? .
A plasmid DNA and a linear DNA (both are of the same size) have one site for a restriction endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one DNA band while linear DNA shows two fragments. Explain.
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.
A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the experiment?
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?
From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
What is the significance of adding proteases at the time of isolation of genetic material (DNA)?
What would be the molar concentration of human DNA in a human cell? Consult your teacher.
While doing a PCR, denaturation' step is missed. What will be its effect on the process?
What would happen when one grows a recombinant bacterium in a bioreactor but forget to add antibiotic to the medium in which the recombinant is growing?
Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
What modification is done on the Ti plasmid of Agrobacterium tumifaciens to convert it into a cloning vector?
For selection of recombinants, insertional inactivation of antibiotic marker has been superseded by insertional inactivation of a marker gene coding for a chromogenic substrate. Give reasons.
A recombinant DNA molecule was created by ligating a gene to a plasmid vector. By mistake, an exonuclease was added to the tube containing the recombinant DNA. How does this affect the next step in the experiment, i.e. bacterial transformation?
Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
Restriction enzymes should not have more than one site of action in the cloning site of a vector. Comment.
Both a wine maker and a molecular biologist who had developed a recombinant vaccine claim to be biotechnologists. Who in your opinion is correct?
A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason?
Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Describe briefly the followings:
(a)Origin of replication
(b)Bioreactors
(c)Downstream processing
Illustrate the design of a bioreactor. Highlight the difference between a flask in your laboratory and a bioreactor which allows cells to grow in a continuous culture system.
