BIOLOGY 3 Marks Question

1. Small volumes of culture can be taken out from the reactor for testing.
2. It has a foam breaker for regulating the foam.
3. It has a control system that regulates the temperature and pH.

1. Palindromic nucleotide sequence is the recognition (specific) sequence present both on the vector and on a desired/alien DNA for the action of the same(specific) restriction endonuclease to act upon.
2. Same restriction endonuclease binds to both the vector and the foreign DNA, cut each of the two strands of the double helix at specific points in their sugar phosphate backbone of recognition sequence for restriction endonucleases/palindromic sequence of vector and foreign DNA, to cut strand a little away from the centre of the palindrome sites, creates overhanging stretches/sticky ends.

1. ampR/ampicillin resistance genes, tetR/tetracycline resistance gene.

They help in identifying and eliminating non-transformants/non-recombinants and selectively permitting the growth of the transformants/recombinants.

2. Simpler process/less cumbersome, in the presence of chromogenic substrate recombinants are colourless, and non-recombinants are blue in colour.

1. Escherichia coli/E.coli.
2. ori.
3. amp^R is the marker gene that helps in identification and elimination of the non transformant growing in ampicillin medium/selectively permitting the growth of the transformant resistant to ampicillin // tet^R is the marker gene that helps in identification and elimination of the non transformant growing in tetracycline medium/selectively permitting the growth of the transformant resistant to tetracyline.

DNA fragments on the agarose gel are negatively charged molecules, and they move towards the anode (The fragments separate according to their size) The separated DNA fragments can be visualised after staining with ethidium bromide.

Followed by exposure to UV radiation Separated fragments are extracted from the gel by elution.

DNA from every tissue from an individual, shows the same degree of polymorphism and is heritable, hence very useful in DNA finger printing.

1. ori - origin of replication.
2. amp^R - ampicillin antibiotic resistant gene.
3. rop - gene to produce the proteins involved in the replication of the plasmid.

Separation/denaturation of two strands of two dsDNA, using two sets of primers/small chemically synthesised oligonucleotides complementary to regions of DNA and (thermostable) DNA polymerase/Taq polymerase, extension of the primers, by enzyme using nucleotides replicates the DNA and if the process of replication is repeated many times multiple copies of DNA are produced.

The following diagram can be considered in lieu of the explanation.

Application = Produces larger biomass leading to higher yields of desired protein/recombinant protein/processing large volume of culture/conversion of raw materials into specific product biologically.

1. Cuts at specific position within the DNA/cuts DNA at specific nucleotide/cuts at palindromic nucleotide sequence.
2. Separation of DNA fragments. (under the influence of electric field)
3. Helps in Identifying and eliminating non-transformants from transformants/selection of transformants.

1. Pst I/Pvu I/EcoR I/Cla I/Hind III/BamH I/Sal I/ Pvu II.
2. ori, rop.
3. amp^R/tet^R.

1. -AATTC / Sticky end.
2. -Ori / Origin of Replication.

2. Pallindromic sequence, because the sequence of base pairs reads same on the two strands when orientation of readigreadingpt the same.
3. PCR - Polymerase Chain Reaction, Importance - amplification of gene of interest (in vitro).

1. a = ampicillin, b = EcoR-I, c = Hind-III, d = tetracycline.
2. Insertion of alien gene into coding sequence of a-galactosidase, results into inactivation of the enzyme// (insertion inactivation), these colonies do not produce any colour (in the presence of chromogenic substrate) hence are identified as recombinant colonies.

Which is isolated from bacterium Thermus aquaticus, and is used to amplify DNA in vitro (by PCR),

It remains active even under high temperature at which DNA denatures.

1. Restriction enzymes EcoRI/Hind II,
2. They recognise and cut at a specific sit on DNA.

1. Restriction enzymes:

1. Restriction enzymes belongs to class of enzymes nucleases which breaks nucleic acids by cleaving their phosphodiester bonds.
2. Since Restriction endonucleases cuts DNA at specific recognition site, they are used to cut the donor DNA to isolate the desired gene.
3. The desired gene has sticky ends which can be easily ligated to cloning vector cut by same restriction enzymes having complementary sticky ends to form recombinant DNA.
4. An example is EcoR1 which is obtained from E.coli bacteria “R” strain which cuts DNA at specific palindromic Recognition site.

5‘ GAATTC 3‘

3‘ CTTAAG 5‘

2. Plasmids:

1. Plasmids are autonomous, extra chromosomal circular double stranded DNA of bacteria.
2. Since they are small and self replicating, they are used as cloning vectors in genetic engineering.
3. Some plasmids have antibiotic resistance genes which can be used as marker genes to identify recombinant plasmids from non recombinant ones.
4. The plasmids are cut and ligated with desired genes and transformed into host cell for amplification to obtain the desired products.
5. An example of artificial modified plasmids are pBR322 ( constructed by bolivar and rodriguez) or pUC (constructed at university at california).

1. Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100 - 1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells.
2. The most commonly used bioreactors are of stirring type. A stirred - tank reactors is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system. pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

1. DNA fragments are negatively charged molecules. Using this property of DNA it is forced to move towards an anode under an electric field through an agarose medium. The DNA fragment separates according to their size through sieving effect provided by agarose gel. Hence the smaller the fragment in size, farther it moves.
2. The DNA fragments after separation are treated with ethidium bromide followed by UV radiation exposure. This enables us to visualize the DNA fragments.

1. Ori-gene: The sequence from where replication start/ any piece of DNA when linked to this sequence can be made to replicate within the host cell, this sequence control the copy number of linked DNA.

2. Antibiotic resistance genes: Help in identifying and eliminating non transformant from transformant/ acts as selectable marker/ helps in ligation of alien DNA at recognition site (present in one of the two antibiotic resistance gene).

3. Rop: Codes for proteins, involved in the replication of plasmids.

Two features that the engineered vectors made to posses are:

1. ORI.
2. Selectable marker.

1. Denaturation: Double-helical DNA is denatured by providing high temperature (95-degree Celsius). DNA polymerase does not get degraded in such high temperatures. The DNA polymerase used in this reaction is thermostable and is isolated from the thermophilic bacteria, Thermus aquaticus (Taq).

2. Annealing: It is the step in which primers are annealed to single-stranded DNA templates. Two sets of primers are used. The temperature of the reaction mixture is lowered to 50-65°C for some seconds to allow annealing of primers. DNA polymerase extends the primer in 5' to 3' direction.

3. Extension: Replication of DNA occurs in vitro.

4. This cycle is repeated several times to generate up to 1 billion identical copies of the DNA.

1. Microinjection.
2. Biolistics/ gene gun.
3. Heat-shock method.
4. Using disarmed pathogen vectors.

1. He would have loaded the samples at the end A. In the wells near the end A.
2.  

• The DNA fragments resolve or separate according to their size, through the sieving effect, provided by the agarose gel matrix.
• The smaller the DNA fragment, the farther it moves and vice-versa.

• He must have stained the DNA with ethidium bromide followed by exposure to UV-radiation.
• he DNA is visible as orange coloured bands.

• Vector is a DNA molecule that can carry a foreign/ desired DNA segment and replicates inside the host cell.
• pBR 322 vector.

• Genes encoding antibiotic resistance are useful as selectable markers because the normal E.coli cells do not carry resistance against any of these antibiotics.
• The ligation of alien DNA is carried out at a restriction site present in one of the antibioticresistance genes, say a foreign DNA is ligated at the BamHI site of tetracycline resistance gene in the vector pBR 322.
• The recombinant plasmids will lose tetracycline resistance due to insertional inactivation.
• The recombinants could be selected out from the non-recombinants by plating the transformants on ampicillin-containing medium.
• The transformants growing on ampicillin-containing medium are transferred to tetracycline-containing medium.
• The recombinants cannot grow on this medium, but non-recombinants will grow on this medium too.

• The DNA formed by combining DNA molecules from different sources/ genomes, is called a recombinant DNA.
• It shows features of those organisms from where the DNAs are isolated.
• The same restriction endonuclease is used to cut both vector DNA and the source DNA at specific locations: hence, the same kind of sticky ends are formed.
• DNA ligase joins together (end-to-end) the complementary cut counterparts by forming hydrogen bonds.

• Bioreactors are the large vessels in which the raw materials are biologically converted into specific products in large quantities, i.e. on commercial scale.
• The bioreactors provide optimum conditions of (i) pH, (ii) substrate concentration, (ii) mineral salts, (iv) vitamins, (v) temperature and (vi) oxygen.

1. Recombinant DNA Technology/ Genetic Engineering.
2. Three steps are:

• Isolation of human DNA with desirable gene.
• DNA segment is incorporated into bacterial plasmid to form recombinant DNA.
• Recombinant DNA is introduced in bacterial cell, which makes pistein directed by human DNA.

1. Any protein encoded gene when expressed in a heterologous host, it is called as recombinant protein.
2. For the large scale production of industrial products.
3. He is curious, inquisitive and having interest in science.

1. The DNA fragments resolve according to their size through the sieving effect of the agarose gel; hence the smaller fragments (in D) have moved farther than those (in C) which are comparatively larger.
2. B is the anode.
3. The matrix provides the sieving effect for the separation of DNA fragments according to their sizes.
4. The separated DNA fragments are stained by ethidium bromide followed by exposure to ultra violet radiation.

• Agrobacterium tumefaciens is a pathogen of several dicot plants.
• It is able to deliver a piece of DNA, called T-DNA into the plant cells and transform them into tumour cells; it dictates the host cells to synthesise its nutrients.
• The Tumour inducing (Ti) plasmid of this bacterium is modified and made non-pathogenic.
• Though it is non-pathogenic, it still has the capacity to deliver its Ti plasmid to the plants and hence, the genes of interest ligated to it; thus it acts as a suitable vector in biotechnology experiments.
• It has been used to transfer nematode-specific genes into tobacco plants, to make them resistant to the nematode, Meloidegyne incognitia, that attacks the roots of tobacco plant.

• Three types- Type I, II and III.
• Because they cut the DNA molecules at a specific site and generate fragments.

1. DNA sample that was loaded on the gel may have got contaminated with nuclease (exo- or endo- or both) and completely degraded.
2. Electrodes were put in opposite orientation in the gel assembly, i.e., anode towards the wells (where DNA sample is loaded).

Since DNA molecules are negatively charged, they move towards anode and hence move out of the gel instead of moving into the matrix of gel.

3. Ethidium bromide was not added at all or was not added in sufficient concentration and so DNA was not visible.
4. After staining with Ethidium bromide it was not observed under UV.