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).