BIOLOGY 3 Marks Question

A reporter gene can be used to monitor the transformation of host cells by foreign DNA.They act as a selectable marker to determine whether the host cell has taken up the foreign DNA or the foreign gene gets expressed in the cell. The researchers place the reporter gene and the foreign gene in the same DNA construct. Then, this combined DNA construct is inserted in the cell. Then, the reporter gene is used as a selectable marker to find out the successful uptake of genes of interest. Example of reporter genes- lac Z gene, which encodes a green fluorescent protein in a jelly fish.

The shake flask method is used for a small-scale production of biotechnological products in a laboratory. whereas stirred tank bioreactors are used for a large-scale production of biotechnology products.
Stirred tank bioreactors have several advantages over shake flasks:

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.

Heat- Denaturation/separation of DNA into two strands.
Primer- Enzyme DNA Polymerase extend the primers using the nucleotides provided in the reaction and the genomic DNA as template.
Thermus aquaticus- source of thermostable DNA polymerase/Taq polymerase.

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.

Correct sequence is-

Gel electrophoresis. DNA are negatively charged, forced to move towards the anode, An electric field in agarose gel matrix, separate according to their size/sieving effect, smaller fragments move faster and further than the larger.

Gel Electrophoresis.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 sequences which are repeated many a times, show a high degree of polymorphism, and form a bulk of DNA in a genome, called as satellite DNA.DNA from every tissue from an individual, shows the same degree of polymorphism and is heritable, hence very useful in DNA finger printing.

If any desired/foreign gene is linked with, Ti plasmid of Agrobacterium tumefaciens, The bacterium is modified into non-pathogenic, Plasmid is cloned into multiple copies, Can delivered into a variety of plants, Desired chemical will be produced.

Restriction endonuclease recognises a specific palindromic nucleotide sequence, in the DNA, Restriction endonuclease cuts the strand of DNA a little away from the centre of palindromic nucleotide sequence but between the same two bases on the opposite strands, leaving single stranded portions at the end/sticky ends.

By providing optimum growth conditions: Temperature, pH, substrate, salts, vitamins, oxygen etc.

EcoRI inspects length of DNA and recognises specific palindromic nucleotide sequence, binds with DNA, cuts each of the two strands of double helix at specific points.

The presence of a chromogenic substrate gives blue coloured colonies, in absence of an insert/in non-transformants, presence of an insert (in the enzyme site), results into (insertional inactivation of the β-galactosidase) colonies which do not produce colour.
Antibiotic resistance method requires duplicate plating/cumbersome procedure.

PCR/polymerase chain reaction.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.

DNA fingerprinting (analysis).
Isolation and digestion of DNA by restriction endonuclease.
Separation of DNA fragments by electrophoresis and transferring them to synthetic membranes/nitrocellulose/nylon.
Hybridisation using labelled VNTR probe.
Detection of hybridised DNA fragments by autoradiography.
Matching banding pattern of DNA/DNA fingerprints/autoradiograms of the passengers killed and that of relatives.

Using Agrobacterium vectors, nematode specific genes introduced into host plant, produced sense - antisense RNA in host cells, ds RNA - initiated RNAi, silenced specific mRNA of nematode, parasite could not survive in transgenic host.

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.  

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).

Palindronic sequence GAATTC,

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.

Bacterium is a source of enzyme Taq polymerase,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.

Plasmids and Bacteriophages are two natural closing vector. Plasmids have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.Two features that the engineered vectors made to posses are:

1. ORI.
2. Selectable marker.

To amplify the gene segment of the interest we should know the sequence of the gene of interest. Primers are designed for amplifying the gene of interest. Two sets of primers (chemically synthesized oligonucleotide stretches) that are complementary to the gene of interest, DNA polymerase enzyme, and deoxynucleotides are added. PCR can then be carried out for its amplification.
PCR consists of 3 steps:

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.

Restriction endonuclease enzyme EcoRI is used in the molecular biology to cut the foreign DNA and vector DNA to form overhangs (called sticky ends). These sticky ends then form hydrogen bonds with their complementary counterparts. The segments with the help of DNA ligases are joined to produce recombinant DNA.

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

Introduction of rDNA into host cell:

1. Microinjection: In this method, the recombinant DNA is directly injected into the nucleus of an animal cell.
2. Gene gun/ Biolistics: In this method, used for plant cells, the cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA.
3. Heat shock method: In this method, the rDNA is forced into the competent cell by incubating the cell with rDNA on ice followed by placing them briefly at 42°C (heat shock) and then putting them back on Ice.

Both. As biotechnology is a very wide area which deals with techniques of using a ‘natural’ organism (or its parts) as well as genetically modified organism to produce products and processes useful for mankind. A wine maker employs a strain-of yeast to produce wine by fermentation (a natural phenomenon), while the molecular biologist has cloned gene for the antigen (that is used as vaccine) in an organism which allows the production of the antigen in large amount.

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.

3.  

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

1.  

E. coli cloning vector pBR322 showing restriction sites (HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI), ori and antibiotic resistance genes (ampR and tetR). rop codes for the proteins involved in the replication of the plasmid.

2. Origin of replication is responsible for controlling the copy number of the DNA sequence inserted.

Since DNA fragments are negatively charged molecules, they can be separated by forcing them to move towards the anode under an electric field through a medium or matrix (usually agarose). This matrix gel acts as sieve and DNA fragments resolve according to their size.

• Ethidium bromide used for staining DNA.
• Stained DNA becomes orange.

• A palindrome in DNA is a sequence of base pairs that reads the same on the two strands, when orientation of reading is kept the same.
• The palindromic sequence, which is recognised by the restriction enzyme, EcoRI is given below, along with the sites where it cuts the DNA strands.

• It is seen that the restriction enzyme cuts the two strands between the same two bases, a little away from the centre of the palindrome sequence.

• 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.

When a specific gene sequence is linked with plasmid vector with the help of DNA ligase, a new combination of circular autonomously replicating DNA is formed and it is called recombinant DNA.
No, recombinant vector can be created only if the same restriction enzyme cuts both the vector DNA and the source DNA, as their palindromic sequences should be same.