BIOLOGY 3 Marks Question

• 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.