BIOLOGY 1.5 Marks Questions

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.

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'

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.

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.

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.

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).

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.

Bands are cut out from agarose gel, extracted from gel piece.

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.

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.

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. ‘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.

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.

• 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.

• 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.

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

• 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.

• 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 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. Denaturation.
2. Annealing.
3. Extension.

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.

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.

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.

1. a- Vector/ plasmid DNA.

2. EcoRI.
3. DNA ligase.

• 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).

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.

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.

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

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.

1. 2¹⁰ molecules = 1024 molecules.
2. YAC vectors.

• 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.

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.

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.
  

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.