Biology 2 Marks Questions

Name of the Restriction enzyme-Bam HI.
The substrate DNA on which it acts-

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-

1. Denaturation of desired double strand DNA-to ssDNA
2. Annealing of primer to ssDNA (single standard).
3. 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.

Plasmid DNA and Chromosomal DNA

Plasmid DNA is an extra-chromosomal DNA molecule in bacteria that is capable of replicating, independent of chromosomal DNA.

Chromosomal DNA is the entire DNA of an organism present inside chromosomes.

Bioreactors: Bioreactors are vessels in which raw materials are biologically converted into specific products, individual enzymes etc. using microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). The most commonly used bioreactors are of stirring type. A biogas plant is a good example of a bioreactor.

Downstream processing: After completion of the biosynthetic stage, the product is subjected through a series of processes before it is ready for marketing as a finished product. The processes include separation and purification, which are collectively referred to as downstream processing. The product has to be formulated with suitable preservatives. Such formulation has to undergo thorough clinical trials as in the case of drugs. Strict quality control testing for each product is also required. Downstream processing and quality control testing vary from product to product.

Enzymes are smaller in size than DNA molecules. This is because DNA contains genetic information for the development and functioning of all living organisms. It contains instructions for the synthesis of proteins and DNA molecules. while, enzymes are proteins which are synthesized from a small strend of DNA known as 'genes', which are involved in the formatoin of the polypeptide chain.

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. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin supports a high copy number.

Some palindromic DNA sequences and the restriction enzymes which act on them are:

1. 5'- AGCT-3' Alul (Arthrobacter luteus) 3'-TCGA-5'
2. 5'-GAATTC-3' EcoRI (Escherichia colt) 3'-CTTAAG-5'
3. 5'-AAGCTT-3' Hindlll (Haemophilus influenzae) 3'-TTCGAA-5'
4. 5'-GTCGAC-3' Sall (Streptomyces albus) 3'-CAGCTG-5'
5. 5'-CTGCAG-3' Pstl (Providencia stuartif] 3'-GACGTC-5'

Chitinase- Chitinase is a enzyme to digest or breakdown glycosidic bonds in chitin cell wall of fungal cell to facilitate its transformation.

No, eukaryotic cells do not have restriction endonuclease because DNA molecules of eukaryotes are heavily methylated. All the restriction endonucleases have been isolated from various strain of bacteria.

RNA and DNA

RNA is a single stranded molecule.	DNA is a double stranded molecule.
It contains ribose sugar.	It contains deoxyribose sugar.
The pyrimidines in RNA are adenine and uracil.	The pyrimidines in DNA are adenine and thymine.
RNA cannot replicate itself.	DNA molecules have the ability to replicate.
It is a component of the ribosomes.	It is a component of the chromosomes.

Exonuclease and Endonuclease

It is a type of restriction enzyme that removes the nucleotide from 5' or 3' ends of the DNA molecule.

It is a type of restriction enzyme that makes a cut within the DNA at a specific site to generate sticky ends.

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.

1. lt restricts foreign DNA from entering normal cell by digesting it at various recognition site. Recognition site is palindromic.
2. They are endonuclease and exonuclease both types.
3. 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.

Restriction nuclease cut DNA at specific sites exonuclease cuts DNA at the ends, endonuclease cuts at specific position within DNA./Restriction endonuclease cuts the DNA at specific pallindromic sequence.

The most commonly used matrix is agarose which is natural polymer extracted from sea weeds. The DNA fragments separate according to their size through sieving effect provided by the agarose gel.

Recombinant DNA technology involves the steps are:
Restriction enzymes, polymerare enzymes, ligases, vectors, and the host organisms.

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of the desired DNA fragment.
4. Amplification of the gene of interest.
5. Ligation of the DNA fragment into a vector using DNA ligase.
6. Transfer of becombinant DNA into the host.
7. Culturing the host cells on a suitable medium on a large scale.
8. Extraction of the desired product.
9. Downstream processing of the product as a finished product ready for marketing.

1. Bacteria is grown in a medium with chromogenic substrate, colonies formed show blue colour no recombinants, no blue colour-presence of recombinants.
2. Gene for the enzyme is inactivated by insertion.

Simple stirred tank bioreactor,
Large scale production of recombinant protein/Raw materials are biologically converted into specific products or enzymes, using microbial plants/animals/human cells.

1. 2 sets of primers, DNA polymerisation/extends the primers using the nucleotides.
2. Thermostable/remain active during high temperature induced denaturation of DNA, Thermus aquaticus.

Lysozyme added to remove the cell wall; Ribonuclease added to remove RNAs; Proteases added to remove proteins, chilled ethanol added to precipitate DNA.

Any protein produced by genetically altered gene in a host.Bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products// A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

Visualised by staining the DNA fragments with ethidium bromide, exposing them to UV radiation (Appear as bright orange bands).Bands are cut out from agarose gel, extracted from gel piece.

1. Meloidegyne incognita.
2. Nematode specific genes isolated cloned and introduced into tobacco plants, ds RNA are produced and RNAi interference initiated, mRNA translation silenced, survival of the nematode not possible in the host plant.

EcoRI.
Eco stands for the genus and species of the prokaryotic cell from which the enzyme was isolated, i.e. E.coli.
R stands for strain.
‘I’ follows order in which enzyme was isolated.

Restriction enzymes cut the strands of the DNA, a little away from the centre of the palindromic sites, but between the same two bases on opposite strands.
They form hydrogen bonds with their complementary cut counterparts.

Ori- Site where replication starts, responsible for controlling copy number.
Restriction site- Site of ligation of alien/foreign DNA, in one of the two antibiotic resistance site/coding sequence of galactosidase.

1. PCR: Polymerase chain reaction

1. PCR is used to alter a particular template sequence for production of newer and desired "DNA by getting sample number of DNA copies.
2. PCR is also used for DNA sequencing. It can be used to detect naturally occurring mutations.
3. It is used in DNA finger printing technique.

2. ELISA {Enzyme-Linked Immuno-sorbant Assay) is an immune chemical test.

1. It is useful in the early diagnosis of diseases using antigen–antibody interactions
2. Used in food industry when detecting potential food allergens.

The two most commonly used bioreactors are simple stirred-tank bioreactor. The importance of using bioreactors is as follows:

1. It provides large volume for cultures. Thus, products are obtained in high quantity.
2. It provides optimal like temperatures and pH for growth of desired product.

1. Exonucleases remove nucleotides from the ends of the DNA, whereas Endonucleases make cuts at specific positions within the DNA.
2. Each restriction endonuclease inspects the DNA molecule in search of a specific recognition sequence. When it gets its specific recognition sequence, it binds to the site and cuts each of the two strands of the double helix at specific points by hydrolysing the phosphodiester backbones.

Linking of DNA fragment is done by DNA ligase/linking of Okazaki fragments or discontinuous synthesis fragments/linking of desired genewith plasmid to form recombinant DNA.

Cohen and Boyer isolated the antibiotic resistant gene, from the plasmid of a bacterium that was resistant to the antibiotic drug, and then linked this gene with the plasmid of Salmonella typhimurium, construction of artificial recombinant DNA molecule.

Restriction enzymes/Polymerase enzymes/Ligase enzymes/Vectors/Host organizms/E.coli/Agrobacterium.

Lymphocytes not immortal/short lived, hence patient requires periodic infusion of such genetically engineered lymphocytes, however if a gene producing ADA is isolated from marrow cells, and introduced into the cells at early embryonic stages it could be a permanent cure.

Thermus aquaticus produces a thermostable, DNA polymerase, when DNA is denatured at high temperature, this enzyme remains active.

(Genetic Engineering Approval Committee) GEAC checks the validity of genetic engineering research and checks the safety of introducing any GMO (genetically modified organisms) because sometimes the GMO results could be unpredictable.

1. ‘a’ - Vector DNA, ‘b’ - Foreign DNA.
2. - EcoRI.
3. - DNA ligase.

1. Plasmid is an autonomously replicating circular extra chromosomal DNA of a bacterium.
2. Adenosine deaminase deficiency, ADA cDNA IS Introduced into these lymphocytes, subsequently returned to the patient Cell could now produce ADA, deficiency overcome, Because these genetically engineered lymphocytes are not immortal.

1. To deliver alien/foreign/desired piece of DNA into the host organism,so that the foreign gene can be amplified,

Example - Plasmids, Viruses.

For maintaining continuous culture system in bioreactors, they are continuously and regularly fed with the growth culture medium steadily, maintaining the appropriate temperature, pressure, pH and other parameters within the bioreactors. From the other end of the bioreactor, finished product generated as a result of microbial action is continuously or periodically withdrawn in specified amounts.
Continuous culture system is maintained in bioreactors because the cells can be maintained in a constant physiological state which ensures continuous and regular outflow of desired products from the bioreactor.

• The bacterial cells are treated with lysozyme, to remove the cell wall.
• The proteins associated with the DNA are removed by treatment with proteases and the associated RNAs are removed by treatment with RNases.
• Similarly other molecules (if any) are removed by appropriate treatments.
• The purified DNA is precipitated by the addition of chilled ethanol.

1. a- Vector/ plasmid DNA.

2. EcoRI.
3. DNA ligase.

This ability to multiply and to produce multiple copies of antibiotic resistance gene E. coli was called cloning of antibiotic resistance gene.

A compound called Ethidium Bromide (EtBr) stains DNA, which on exposure with ultraviolet radiations gives orange light. Hence, DNA fragments appear as orange bands.

Bacterium Thermus aquaticus is a source of enzyme tag polymerase, As it is a thermostable enzyme and work at high temperature, it is used to amplify DNA in vitro by PCR. The amplified fragment of desired DNA can be used to ligate with the vector for further cloning

T-DNA is the only essential part required to make Ti plasmid a cloning vector. The plasmid is disarmed by deleting the tumour inducing genes in the plasmid so that it becomes an effective cloning vector and remove it harmful effect.

Downstream processing:

• All the processes to which a product is subjected to before being marketed as a finished product are called downstream processing.
• It includes:

1. Separation of the product from the reactor.
2. Purification of the product.
3. Formulation of the product with suitable preservatives.
4. Quality control testing and clinical trials in case of drugs.

If denaturation of double-stranded DNA does not take place, then primers will not be able to anneal to the template, no extension will take place, hence no amplification will occur.

Biomolecules do not exhibit biological activity in anhydrous conditions. DNA may get damaged under anhydrous condition but has the ability to repair later on. Protein molecule may get denatured under anhydrous conditions.

• The specific DNA sequence, where the replication of DNA is initiated, is called origin of replication (Ori).
• For the multiplication of the alien DNA in the host, it has to be integrated to the origin of replication (Ori).

If the restriction enzymes have more than one recognition site in a vector, than the vector itself will get fragmented on treatment with the restriction enzyme.

The separated band of DNA (after gel electrophoresis) are cut out from agarose gel and DNA is extracted from the gel piece. This step is called elution.

Selectable marker helps in identifying and eliminating non-transformant DNA and in selectively permitting the growth of transformants. In the absence of a selectable marker, it will not be possible to differentiate between transformants and non-transformants. Thus, carrying the experiment to its logical end would be impossible in the absence of selectable marker.

1. DNA fragments in band D are smaller than those of band C.

• The DNA fragments separate according to their size through the sieving effect provided by the gel; hence the smaller fragments move farther away than the larger ones.

2. End towards B.
3. The gel containing DNA fragments is stained with ethidium bromide and exposed to UV radiation; orange-coloured bands (of DNA) become visible.

1. Restriction endonucleases: for cutting the desired DNA at desired places.
2. Gel electrophoresis: for separating the desired DNA fragments.
3. Ligase enzyme: for creating recombinant DNA molecule.
4. DNA delivery system: like electroporation, microinjection, gene gun method.
5. Competent host (usually bacteria/ yeast): to take up recombinant DNA.

• Bacterial colonies with cloning vector A are colourless, as they are recombinants with the insert.
• Presence of the insert has resulted in insertional inactivation of the enzyme and hence, do not produce any colour.
• Bacterial colonies with cloning vector B are non recombinants, i.e. have no insert and produce colour as the enzyme is produced.

If the restriction enzymes would cut DNA at random sites instead of at specific sites, then the DNA fragments obtained will not have ‘sticky ends’. In the absence of sticky ends, construction of recombinant DNA molecule would not be possible.

In a gene cloning experiment, first a recombinant DNA molecule is constructed, where the gene of interest is ligated to the vector and introduced inside the host cell (transformation).
Since, not all the cells get transformed with the recombinant/plasmid DNA, in the absence of selectable marker, it will be difficult to distinguish between transformants and non-transformants, because role of selectable marker is in the selection of transformants.

In the enzyme Hind III; ‘H in’ refers to Haemophilus influenza, D refers to the strain of H. influenza and III refers to the sequence in which this enzyme was discovered.

The two methods of vectorless gene transfer are:

1. Micro-injection: The technique of introducing foreign gene in a target cell by injecting the DNA, directly into the nucleus, by micro-needle is called micro-injection.
2. Electroporation: It is the process in which transient holes are produced in the plasma membrane of the target cell, to incorporate foreign DNA.

Lysing enzymes are used to open up the cell to obtain DNA along with other macromolecules for genetic experiments. Bacterial cells are treated with lysozyme, plant cells are treated with cellulase, and fungal cells are treated with chitinase for lysing.

1. Two primers are required:

• The primers become annealed at the complementary regions of the DNA strands, that have been separated by denaturation; it makes the extension (synthesis of new DNA strands easy).
• DNA polymerase extends the primers using the nucleotides is the medium on the genomic template.

2. The source is Thermus aquaticus: The Taq polymerase is thermostable and withstands the high temperature used in denaturation.

1. Denaturation.
2. Annealing.
3. Extension.

In PCR, because it is a thermostable DNA polymerase enzyme, gets isolated from bacteria Thermus aquaticus from hot water springs, and it does not get denatured at high temperature which is required during PCR and works as normal DNA polymerase enzyme (whereas the normal DNA polymerase gets denatured at high temperature).

1.  

Such sequences are called palendromes in DNA.

2. Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA and cuts the DNA strands a little away from the centre of the palindromic sequence, but between the same two bases on the two strands.

• This leaves single-stranded portions, called sticky ends, overhanging at the end of each strand.
• Since, the stickiness facilitates the action of DNA ligase, they easily form hydrogen bonds with their complementary counterparts.

Role of proteases is to degrade the proteins present inside a cell (from which DNA is being isolated). If the proteins are not removed from DNA preparation then they could interfere with any downstream treatment of DNA (such action of restriction endonuclease, DNA ligase etc).

1. The air bubbles dramatically increase the surface area for oxygen transfer.
2. Separation and purification.

PCR is a very sensitive technique which enables the specific amplification of desired DNA from a limited amount of DNA template.
Hence, it can detect the presence of an infectious organism in the infected patient at an early stage of infection (even before the infectious organism has multiplied to large number).

It is added to precipitate the purified DNA to isolate it.

Both are right because 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.

rDNA	cDNA
It is the DNA which is formed by joining together the DNA from two different organisms.	It is the DNA which is obtained from RNA template catalysed by reverse transcriptase enzyme.

Gene cloning refers to a process in which a gene of interest is ligated to a vector. The recombinant DNA thus produced is introduced in a host cell by transformation. Each cell gets one DNA molecule and when the transformed cell grows to a bacterial colony, each cell in the colony has a copy of the gene.

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

Yes, for the multiplication of the alien DNA in the host, it has to be integrated to the origin of replication. The host cell is not competent, hence it is not picking up the foreign DNA.

rDNA is the plasmid vector containing the foreign DNA whereas recombinant protein is the product of transgenic gene in the host body or cell.

The experiment will not likely be affected as recombinant DNA molecule is circular and closed, with no free ends.
Hence, it will not be a substrate for exonuclease enzyme which removes nucleotides from the free ends of DNA.

The cell or organism derived from the same parents by asexual mean which are genetically identical to each other and to the parent are called clones.

• The prokaryotes/ bacteria have restriction enzymes to restrict the growth of bacteriophages, which infect them; it is a defence mechanism of bacterial cells.
• They do so by cutting the DNA of the phage with restriction enzyme.
• Bacteriophages do not infect eukaryotes, eukaryotes have other defence mechanisms.

Foreign gene is usually ligated to a plasmid vector and introduced in the host. As plasmid replicates and makes multiple copies of itself, foreign gene also gets replicated and it's copies are also made. When the host organism divides, its progeny also receives the plasmid DNA containing the foreign gene.

If the restriction enzymes have more than one recognition site in a vector, then the vector itself will get fragmented on treatment with the restriction enzyme.

No, as exonuclease acts on the free ends of linear DNA molecule. Therefore, instead of producing DNA fragments with sticky ends, it will shorten or completely degrade the DNA fragment containing the gene of interest, and the circular plasmid (vector) will not get cut as it lacks free ends.

The ability of the bacterial cell to take up DNA through pores in cell wall is called competent. DNA is a hydrophilic molecule and hence, it cannot pass through the cell membrane. So, cell is made ‘competent’ by treating with suitable divalent ion; like calcium.

Genetic engineering is the manipulation of genetic materials which can be introduced into host organisms and thus change the phenotype of the host organism. The natural genetic engineer of plant is Agrobacterium tumifaciens.

It is the vector to transform a foreign gene.

High-copy number, because higher the copy number of vector plasmid, higher the copy number of gene and consequently, protein coded by the gene is produced in high amount.

It is the set up to culture large volumes of transgenic bacteria to get large quantities of the product protein.

• EcoRI is the restriction endonuclease obtained from Escherichia coli RY 13.
• EcoRI is a restriction endonuclease which cuts the DNA strands at specific sites within the DNA.
• Exonucleases remove the nucleotides or bases from the ends of the DNA strands.

The cell is made competent by the following methods:

1. Chemical method:

• The cell is treated with specific concentration of a divalent cation such as calcium to increase pore size in cell wall.
• The cells are incubated with recombinant DNA on ice, followed by placing them briefly at 42°C and then putting it back on ice. This is called heat shock treatment.
• The bacteria now take up the recombinant DNA.

2. Physical methods The physical methods include:

• Micro-injection method: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics or gene gun method: Cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in plants.

Agrobacterium tumifaciens is a pathogen of several dicot plants which exhibit natural genetic engineering in plant.Reasons:

1. It is able to deliver a piece of DNA called ‘T-DNA’ to transform normal plant cell into a tumour cell.
2. The DNA transforms the normal cells into tumour cells which direct them to produce the chemical essential for the pathogen.

As this process occurs in nature it is called natural genetic engineering.

A bioreactor provides optimum growth conditions like temperature, pH, substrate, salts, vitamins and oxygen to get desired products.

• Stanley Cohen and Herbert Boyer.
• Salmonella typhimurium.
• It was possible with a restriction endonuclease.

Agrobacterium tumifaciens is a soil bacterium which causes disease in many dicot plants. It is able to deliver a piece of DNA known as T-DNA, to transform the normal cells into tumour cells and direct these tumour cells to produce the chemicals required by the pathogen. The tumour inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to the plants but still deliver genes of interest into a variety of plants.

1.  

• When an alien DNA is inserted within the coding sequence of the enzyme, it results in inactivation of the enzyme.
• The recombinants can be differentiated from the non-recombinants by their inability to produce colour in the presence of a chromogenic substrate.
• The recombinants do not produce any colour, while the non-recombinants produce a blue colour with the chromogenic substrate in the medium.

2. Since, the insert inactivates the enzyme, β-galactosidase, this method is called insertional inactivation.

DNA being hydrophilic in nature cannot pass through the cell membranes into the host. Therefore, cloning vectors are required to transfer the DNA into the host by attaching the desired DNA to it.
The two cloning vectors that are used are plasmids and bacteriophages.

In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing the gene of your interest). Since, maintaining a high copy number of plasmids is a metabolic burden to the microbial cells, it will thus tend to lose the plasmid.

The given bioreactor shown in the figure is the simple stirred-tank type bioreactor.
Its purpose is large scale Production of recombinant protein enzymes, using microbial plants, animals and human cells.

No, as exonuclease acts on the free ends of linear DNA molecule. Therefore, instead of producing DNA fragments with sticky ends, it will shorten or completely degrade the DNA fragment containing the gene of interest, and the circular plasmid (vector) will not get cut as it lacks free ends.

In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing the gene of your interest). Since, maintaining a high copy number of plasmids is a metabolic burden to the microbial cells, will thus tend to lose the plasmid.

S.No	Simple stirred-tank bioreactor	Sparged stirred-tank bioreactor
1.	A simple stirred-tank bioreactor is usually cylindrical or with a curved base to facilitate even mixing of reactor contents.	The sparged-stirred-tank bioreactor also facilitates the mixing of components and ensures oxygen availability throughout the bioreactor.

1. Origin of replication/ ori site: From here the replication starts (and any piece of DNA when linked, can be made to replicate within the host cell).
2. At least two Selectable markers: Helpful in identifying and eliminating non-transformants.
3. Unique Restriction sites for more than one restriction enzymes: The foreign DNA links to this region of the plasmid.

A compound called Ethidium Bromide stains DNA, which on irradiating with Ultraviolet, fluoresce gives orange light. Hence, DNA fragments appear as orange band in the presence of Ethidium Bromide and UV.

It is used to inject the foreign gene into a host cell, directly.

A circular DNA opens up to resemble a single linear DNA. A Linear DNA is divided into two fragments after cleavage. Hence, circular DNA shows one band, while linear DNA shows two bands.

1. The process of cutting the desired gene (also called restriction digestion) is carried out by incubating the purified DNA molecules with the specific restriction enzyme at optimal conditions of pH, temperature etc. of the enzyme.
2. The stirrer facilitates the even mixing and oxygen availability throughout the contents in the reactor.