BIOLOGY 5 Marks Questions

• Identifying the restriction endonuclease that recognises the palindromic nucleotide sequence of the desired gene.
• The restriction endonuclease inspects the DNA sequences - finds and recognises the site.
• Cuts each of the double helix at the specific point - a little away from the centre of the palindromic site - between the same two bases on the opposite strand
• Makes the over hanging stretch single stranded portion as a sticky end.

• By PCR/Polymerase Chain Reaction.
• Desired gene is synthesised in vitro.
• DNA is denatured.
• Annealed using two sets of primers.
• Thermostable Taq polymerase extends the primers using nucleotides (provided in the reaction and genomic DNAas template).
• Amplified fragments are ligated.

• Should have ori/origin of replication,
• Has selectable marker, genes encoding for an antibiotic resistance/genes encoding for a - galactosidase,
• Has cloningsite/recognition site, for the restriction enzyme to recognise.

2. DNA is a hydrophilic molecule

Bacterial cell is made competent by treating with specific concentration of Ca++ ion/divalent ions, incubating the mon ice, heat shock for a short period and placing it back on ice again.

1. A vector with two antibiotic resistance markers.
2. Preparation of two kinds of media plates, with one antibiotic each.

Transformed cells are first plated on the antibiotic plate which has not been insertionally inactivated (say, ampicillin) and incubated overnight for growth of transformants. For selection of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline) plate (which got inactivated due to insertion of gene). Non-recombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only on ampicillin plate.

This entire exercise is laborious and takes more time (two overnight incubation) as well. However, if we choose insertional inactivation of a marker that produces colour in the presence of a chromogenic compound, we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.

Structure of Bioreactor:

• It is a cylindrical structure with a curved base.
• A stirrer is present for even mixing and oxygen availability throughout the reactor.
• There is an agitator system, an oxygen delivery system, a foam control system, a temperature control system, etc.
• There is a sampling port through which small volumes of culture can be taken out periodically.

A flask in a laboratory cannot be used for producing recombinant DNA on large scale. Unlike a bioreactor; a flask cannot be used to grow culture continuously.

Difference:

1. Amplification of gene of interest using PCR is required.
2. Denaturation, annealing and extension are the basic steps of PCR.
3. Kary Mull is developed PCR in 1985.
4. Honesty and sense of responsibility towards his duty.

1. Plasmid.
2. Cosmid.
3. Bacteriophage lambda.
4. BAC.

1. Restriction enzymes are derived from a defence unit called restriction modification system which protects the microorganisms from harm.
2. They are called as molecular scissors because they cut DNA molecules at specified location indicated by presence of specific recognition sequence.
3. No, the strands cut from within are then joined by another enzyme called DNA ligase. This joining is either with self or with another different strand.
4. Varun is curious, has scientific temperament and asks quick and intelligent questions.

1. Palindromes.
2. Restriction endonucleases.
3. EcoRI.
4. Sticky/ overhanding strands.

• RNA is removed by treatment with ribonuclease and proteins are removed by treatment with protease.
• After several treatments, the purified DNA is precipitated by adding chilled ethanol.
• The bacterial/ plant/ animal cell is broken down by enzymes to release DNA, along with RNA, proteins, polysaccharides and lipids.
• Bacterial cell is treated with enzyme lysozyme.
• Plant cell is treated with enzyme cellulase.
• Fungal cell is treated with chitinase.

Cutting of DNA at specific locations:

• The DNA is cut using restriction enzymes.
• The purified DNA is incubated, with the specific restriction enzyme at conditions optimum for the enzyme to act. Isolation of desired DNA fragment
• Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked.
• Since the DNA is negatively charged, it moves towards the positive electrode or anode and in the process, DNA fragments separate out based on their sizes.
• The desired DNA fragment is eluted out.

Amplification of gene of interest using PCR:

• The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro. 

1. Host cells are incubated with rDNA on ice.
2. Followed by placing them briefly at 42°C.
3. Then transfer them back on ice.

This enables the host cells (bacteria) to take up the rDNA.

• Sequence Annotation-total DNA from a cell is isolated, converted into random fragments of relatively smaller sizes, and cloned in suitable host using specialized vectors.
• The cloning resulted into amplification of each piece of DNA fragment.
• The fragments were sequenced using automated DNA sequencers, these sequences are then arranged based on some overlapping regions (present in them).
• This requires generation of overlapping fragments (for sequencing).
• Specialised computer based programmes were developed, and these sequences were subsequently annotated and assigned to each chromosome.

1. Restriction endonucleases.
2. Agarose gel.
3. DNA - ligase.
4. Plasmid.
5. Hind II.
6. Elution.

1. Engineered vectors help easy linking of foreign DNA.
2. They facilitate selection of recombinants from non-recombinants.

2. Features of Cloning vectors:

1. Origin of Replication (Ori):

• This is the sequence of DNA from where replication starts.
• Any piece of alien/ foreign DNA linked to it is made to replicate within host cell; it also decides the copy number of the linked DNA.

2. Selectable marker: A marker is a gene, which helps in selecting the host cells, which are transformants/ recombinants from the non-recombinant ones, eg ampicillin and tetracycline resistant genes in E.coli

3. Cloning site: The vector should have a few, preferably one unique recognition site to link the foreign DNA, presence of a particular cloning/ recognition site enables the particular restriction enzyme to cut the vector DNA.

• The purified DNA molecule containing the desired gene is incubated with the specific restriction endonuclease, at optimal conditions of pH, temperature, etc. of that enzyme.
• Similarly, the vector DNA is also incubated with the same restriction endonuclease under optimal conditions.
• This results into cutting of the DNA into fragments.

• Gel electrophoresis is used to separate the DNA fragments produced by restriction digestion.
• The DNA fragments resolve according to their sizes, through the sieving effect provided bythe agarose gel.

3. The resultant fragments are joined to the vector by use of DNA-ligases.

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

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

1. DNA template: The double-stranded DNA that needs to be amplified.

2. Primers: These are chemically synthesized oligonucleotides (short segment of DNA) that are complementary to the regions of DNA template.

3. Enzymes: Two commonly used enzymes in PCR reaction are:

• Taq Polymerase: It is isolated from a thermophilic bacterium, i.e. Thermus aquaticus and has a property to remain active during the high temperature induced denaturation of double-stranded DNA. It also helps in the amplification of a segment of DNA.

• Vent Polymerase: (isolated from Thermococcus litoralis).

4. Nucleotide bases: These are added by DNA polymerase to the growing chain. Three main steps involved in the PCR technique are:

Step I Denaturation: The double-stranded DNA is denatured by using high temperature of 95°C for 15 seconds. Now each separated single strand acts as a template for DNA synthesis.

Step II Annealing: Two sets of oligonucleotide primers are annealed (hybridised) to the separated single-strands. This step is carried out at a slightly lower temperature (40-60°C).

Step III Extension: The thermostable enzyme Taq DNA polymerase is used in this reaction, extends the primers by adding dNTPs (deoxynucleoside triphosphates) complementary to those of the template DNA. Mg²⁺ is required as a cofactor for thermostable DNA polymerase.