BIOLOGY 2 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).

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.

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. 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.