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.

• If a foreign DNA is ligated at the BamH I site of tetracyclin-resistance gene in the pBR 322, the recombinant plasmid will lose the tetracycline-resistance.
• The transformant can be selected out from the nontransformants by culturing on a medium containing ampicillin.
• The transformants are transferred to a culture medium containing tetracyclin; there they fail to grow but non-transformants grow in tetracyclincontaining medium as well as ampicillin-containing medium.
• Here, one antibiotic gene facilitates selection of transformants, while the other antibiotic gene gets inactivated by the insertion of alien DNA and facilitates selecting the recombinants.

1.  

• Restriction endonucleases are used to cut the DNA strands of vector and the source/ foreign DNA at specific locations, containing the genes of our interest).
• When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of sticky ends, which are easily joined by a DNA ligase.

2.  

• 'Ori' is the sequence of nucleotides in a DNA, where replication starts.
• When a piece of alien DNA is ligated to it, it replicates and forms multiple copies (cloning) within the host.

3.  

• Gel electrophoresis is used to separate the DNA fragments formed by the restriction digestion.
• The separated DNA fragments are isolated and used in biotechnological experiments.

• 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.

A bacterium Agrobacterium tumefaciens has T-DNA in its Ti-plasmid which is able to transform normal plant cells to tumour cell and cause crown gall tumours in plants.
In animals retroviruses have this ability.

Features of Cloning Vector There are certain features that are required to facilitate cloning into a vector. These are:

1. Origin of replication (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.
2. Selectable marker: It helps in identifying or selecting transformants and eliminating non-transformants by selectively permitting the growth of the transformants. 'Transformation is a procedure, through which a piece of DNA is introduced into the host bacterium. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered as useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.
3. Cloning (recognition) sites: These are generally required to link the foreign or alien DNA with the vector. For this, the vector requires very few or single recognition sites for commonly used restriction enzymes. If more than one recognition sites is present within the vector, it will generate several fragments that will lead to more complication in gene cloning.

Vectors for cloning genes in plants and animals: In plants, Agrobacterium tumefaciens (a pathogen of several dicot plants) is able to deliver a piece of DNA known as 'T-DNA' (transfer-DNA) to transform normal plant cells into tumour cells to produce chemicals required by pathogen. This is done as the tumour inducing (Ti) plasmid of Agrobacterium tumefaciens has been modified as a cloning vector which is no longer pathogenic to plant but is still able to deliver genes of interest. Similarly, retrovirus, adenovirus, papilloma virus are also used as a cloning vectors in animals because of their ability to transform normal cells into cancerous cells.

• 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.

Agriculture, Medicine, Food industry and genetic engineering are the some areas.

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.

Vectors for cloning genes in plants and animals In plants, Agrobacterium tumefaciens (a pathogen of several dicot plants) is able to deliver a piece of DNA known as T-DNA' (transfer-DNA) to transform normal plant cells into tumour cells to produce chemicals required by pathogen. This is done as the tumour inducing (Ti) plasmid of Agrobacterium tumefaciens has been modified as a cloning vector which is no longer pathogenic to plant but is still able to deliver genes of interest. Similarly, retrovirus, adenovirus, papilloma virus are also used as a cloning vectors in animals because of their ability to transform normal cells into cancerous cells.

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.

1. Plasmid DNA: It is often used for constructing recombinant DNA, by ligating the gene of interest with it; it is used as the cloning vector.

2. Recognition sequences: These are the sequences of base pairs in DNA, which a restriction enzyme recognises and cuts the DNA
3. Gel electrophoresis: It is used to separate the DNA fragments, which separate/ resolve according to their size through the sieving effect of the gel; the DNA is isolated from the gel by the process of elution.

• When an alien DNA or recombinant DNA is ligated within the coding sequence of an enzyme, the enzyme becomes inactivated; this phenomenon is called insertional inactivation.
• A recombinant loses the ability to produce a (blue) colour with a chromogenic substrate; hence, it can be distinguished from the non-recombinants, which produce blue-coloured colonies with the chromogenic substrate.
• This method is preferred to the antibiotic-resistance method as the latter is a cumbersome procedure that requires simultaneous plating on two plates having two different antibiotics.

• 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.

1. The vector DNA is cut at a particular restriction site using a restriction enzyme (to cut the desired DNA segment). The alien DNA is then linked with the plasmid DNA using an enzyme called ligase to form the recombinant vector.
2. A restriction enzyme recognizes and cuts the DNA at a particular sequence, called recognition site. If more than one restriction enzymes are present, they will generate several segments and will complicate gene cloning.

Agrobacterium tumafaciens harbours a mega plasmid called Ti-plasmid.
This has a T-DNA region flanked by left border and right border sequence. The T-DNA gets transferred and integrates with the host plant DNA. This property of Ti-plasmid has been exploited for cloning of gene of interest and stably integrating them in the plant genese. Therefore, by using Ti-plasmid or its derivatives, recombinant plant cells with desired genes of interest stably integrated in the plant genome has been successfully produced.

Features of Cloning Vector There are certain features that are required to facilitate cloning into a vector. These are:

1. Origin of replication (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.
2. Selectable marker: It helps in identifying or selecting transformants and eliminating non-transformants by selectively permitting the growth of the transformants. 'Transformation is a procedure, through which a piece of DNA is introduced into the host bacterium. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered as useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics.
3. Cloning (recognition) sites: These are generally required to link the foreign or alien DNA with the vector. For this, the vector requires very few or single recognition sites for commonly used restriction enzymes. If more than one recognition sites is present within the vector, it will generate several fragments that will lead to more complication in gene cloning:

The steps are:

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Amplification of the desired DNA fragment.
5. Ligation of the DNA fragment into a vector.
6. Transferring the recombinant DNA into the host.
7. Culturing the host cells and extraction of the desired gene product on a large scale.
8. Downstream processing, i.e. separation and purification.

1. Cloning sites are the recognition sites in the vector DNA, for the restriction enzymes; these are the sites where specific restriction enzymes cut the DNA strands.
2. When an alien DNA is ligated at a particular cloning/ recognition site, the recombinant plasmid loses its function of the gene, where the recognition sequence is present; for example if an alien DNA is ligated at a recognition site of an antibiotic resistance gene in E.coli, the recombinant will lose the resistance to the particular antibiotic.
3. The restriction sites in pBR 322 are:

• BamH I site of tetracyclin-resistance gene.
• Sal I site of tetracyclin-resistance gene
• Pst I site of ampicillin-resistance gene.

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of desired DNA fragment.
4. Ligation of DNA fragment into vector.
5. Transferring recombinant DNA into host.
6. Culturing host cells at large scale.
7. Extraction of the desired product.

Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. The injected DNA is selectively replicated and expressed in the host bacterial cell resulting in a number of phages which burst out of the cell and reinfect neighbouring cells. Their ability to transfer DNA from the phage genome to specific bacterial hosts during the process of bacterial infection give it to the property be used as vectors.
Examples are: Phage lambda and M13 phages.

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

The reasons that could be possible are as follows:

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.

• Polymerase Chain Reaction.
• Denaturation, Annealing and Extension.

1. 5' - GAATTC - 3'

2. EcoRI is the restriction endonuclease that recognizes this palindrome.
3. When the restriction enzyme cuts the DNA strands a little away from the centre of the palindromic sequence, between the same two bases on both the strands, sticky ends are produced.

• The stickiness of the ends facilitates the action of the enzyme DNA-ligase.

1. In the above bacterial cell, i.e. A is plasmid B is chromosomal DNA. Plasmid is used as a vector in rDNA technology.
2. If the enzyme EcoRI acts on both linear DNA and plasmid DNA, each having three recognition sites, the restriction enzyme will generate 3 fragments from plasmid DNA (as it is circular) and 4 fragments from linear DNA.

1. Positive terminal- ‘B’

Negative terminal- ‘A’

1. DNA is negatively charged. Because of its negative charge, DNA moves towards the positive electrode (anode).
2. The separated DNA fragments are separated by elution. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.

• Taq polymerase.
• It is isolated from Thermus aquaticus bacterium.

DNA is a hydrophillic molecule so it is difficult to cross lipid molecules of membrane.

• To make E. coli cells competent to take up DNA, they should be treated with divalent cations or calcium.
• In 1970, Mandel and Higa found that E.coli cells become competent to take up DNA when they are suspended in cold calcium chloride.

• By treating bacteria with cold calcium chloride or lysozyme.
• Escherichia coli, Bacillus subtilis.

Naming of restriction enzymes:

• The first letter of the name comes from the genus of the bacterium.
• The second and third letters come from the name of the species of the prokaryote cell from where it is isolated.
• The next letter comes from the strain of the prokaryote.
• The Roman Numbers following these four letters indicate the order in which the enzymes were isolated from that strain of the bacterium, e.g. EcoRI is isolated from E.coli, RY 13.

1.  

• Taq polymerase is a thermostable enzyme that can withstand the high temperature used in the denaturation step, whereas ordinary DNA polymerase cannot withstand high temperature.
• It is obtained from the bacterium, Thermus aquaticus.

2. PCR is used for gene amplification, i.e., to make multiple copies of a gene or a DNA segment.

• This enzyme is used to prevent unwanted self-ligation of vector DNA molecule.
• Sources: from bacteria (BAP), from calf intestine (CAP)

1. The recombinant/ transformaot can be selected out from the non-recombinants/ non-transformants by Plating the transformants on ampicilin-containing medium. The transformants will grow in it, while the non-transformants will not grow.
2. It acts as a selectable marker.

1.  

1. DNA is a hydrophilic molecule that cannot pass through cell membrane; so the bacterial cells must be made competent to take up the DNA.
2. The bacterial cells are made competent by treating them with a specific concentration of a divalent cation, such as calcium; this increases the efficiency with which DNA enters the bacterium.

2.  

1. Biolistics/ Gene gun.
2. Micro-injection.

Restriction Enzymes (Molecular scissors): These are enzymes which are used for cutting of DNA at specific locations during DNA technology. These enzymes belong to a larger class of nucleases, which are of two types

1. Exonucleases: remove nucleotides from the ends of the DNA (either 5' or 3’) in one strand of duplex.
2. Endonucleases: make cuts at specific position within DNA. The first restriction endonuclease named Hind II was isolated by Smith Wilcox and Kelley in 1968.

It was found that it always cuts DNA molecule at a particular point recognising a specific sequence of the six base pairs known as the recognition sequence for Hind II.
More than 900 restriction enzymes are known today that have been isolated from over 230 bacterial strains and each of which recognises different recognition sequences. Restriction endonucleases are not present in eukaryotic cells.

1. A bioreactor is used for obtaining a desired gene product in large quantities by processing large volumes of cultures of microbial, plant or animal cells.
2. The stirring type of bioreactors (Simple stirred-tank bioreactor or sparged stirred-tank bioreactor) are commonly used.
3. The components of simple stirred-tank bioreactor include:

• A stirrer.
• Flat-bladed impeller.
• An oxygen delivery system.
• A foam control system.
• pH control system.
• Sampling port.

1. Palindromic sequence and the site where EcoRI cuts it (indicated by the arrows).

2. Sticky ends

1. He would have loaded the samples near end A; in the wells.
2. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
3. After staining the DNA with ethidium bromide followed by exposure to UV radiations the DNA bands appear coloured.

1. Biotechnology deals with the techniques of using live organisms or enzymes from organisms to produce products and processes useful to human.
2. These are formed by integration of a segment of DNA of the bacterium Agrobacterium tumifaciens into the host DNA.
3. Rahul's uncle has a positive desire to know about latest discoveries and developments in the field of science.

1. A - Denaturation process
2. B - Primers
3. C - Taq DNA polymerase. Tag polymerase is a thermostable enzyme, which remains active during the high temperature and induces denaturation of DNA,

1. Selectable marker in the cloning vector helps to identify and eliminate the non-recombinants and to selectively permit the growth of recombinants.
2. Ori (origin of replication) is a sequence of DNA from where replication starts; any piece of alien DNA ligated to this can be made to replicate within the host cut and it determines the copy number too.
3. Rop codes for the proteins involved in the replication of the plasmid.

The first letter ‘H’ indicates the genus of the organism, from which the enzyme was isolated, i.e. H-genus, Haemophilus. The roman number (III) denotes the sequence in which the restriction endonuclease from that particular genus, species and strain of bacteria have been isolated, i.e. third restriction endonuclease to be isolated from this species.

• The DNA fragments are separated by electrophoresis using agarose as the matrix.
• Since, DNA fragments are negatively charged, they move towards the anode under the electric field through the medium and separate/ resolve according to their size due to the sieving effect of agarose gel.
• The separated fragments can be viewed by staining the DNA with ethidium bromide followed by exposure to UV radiation.
• Elution is the process in which the separated bands of DNA are cut out from the gel and extracted.

Three uses of PCR are:

1. It is used to during IDNA for production of newer and desired DNA.
2. It is used for DNA sequencing.
3. It is used in DNA fingerprinting.

When a DNA molecule is created by ligated a gene to a plasmid vector; it becomes a circular DNA which is ready to replicate in host organism. After this stage, addition of exonuclease is not going to affect the process because the DNA does not have a free end and hence enzyme exonuclease will not get a substrate to show its action. So, in this experiment; bacterial transformation is not going to be disturbed.

1. It helps in identifying or selecting transformants and eliminating non-transformants by Selectively permitting the growth of the trarsformants.
2. 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.
3. Rop codes for the protein involved in the replication of the plasmid.

1. The protein coded by a recombinant gene in the transgenic or genetically modified organism, is called a recombinant protein.
2. This can be harvested on a large scale by culturing the transgenic cells in large volumes in bioreactors.
3. The precautions include:

• The optimum temperature must be maintained.
• There should be foam control.
• Suitable pH must be provided.

1. A- Vector DNA,/ Plasmid DNA; B- Foreign DNA.
2. 5'- GAATTC - 3'; 3'-CTTAAG - 5'
3. DNA ligase.

Application: These bioreactors are used to produce large quantities of products enzymes, etc., using microbial, plant, animal or human cells.

• In gel electrophoresis, the molecules resolve according to their molecular size by the sieving effect provided by the gel.
• DNA being negatively charged, moves towards the anode in the electric field.
• Use of DNA isolated:

1. The DNA fragments may be used to construct recombinant DNA by joining them with cloning vectors.
2. The desired DNA fragments may be amplified, i.e. making multiple copies, by polymerase chain reaction (PCR).

• The restriction enzyme, ECORI recognises the palindromic sequence, shown below:

• It cuts the DNA strands a little away from the centre of the palindrome site, but between the same two bases on both the strands (indicated by the arrows)
• This leaves single-stranded stretches, called sticky ends, overhanging at the end of the strands.

• DNA ligase can catalyse hydrogen bond formation between the cut complementary parts of the vector DNA and foreign DNA.