Biotechnology: Principles and Processes

PCR proceeds through: Denaturation of the DNA strands (C) → Annealing of primers to the single-stranded DNA (A) → Treatment with Taq polymerase and deoxynucleotides for primer extension (D) → Extension of the new strand (E) → Amplification of the DNA (~1 billion times) after repeated cycles (B). Order: C → A → D → E → B.

Insertion of recombinant foreign DNA into the coding region of $\beta$-galactosidase causes insertional inactivation. Non-recombinants retain active enzyme producing blue colonies with X-gal, while recombinants cannot produce active enzyme and appear white.

DNA fragments separated by gel electrophoresis cannot be seen directly; the gel must first be stained with a compound such as ethidium bromide, and the DNA bands are then visualised under UV radiation. Pure DNA fragments cannot be seen in visible light without staining, so the statement in option (a) is incorrect.

In somatic hybridisation, protoplasts (cells whose cell walls have been removed) from two different varieties of plants are fused to form hybrid protoplasts. These are then grown to form a new plant (somatic hybrid), e.g. pomato.

Bioreactors are used to produce large quantities (100–1000 litres) of bacterial cultures, not small-scale cultures – so this statement is incorrect. Bioreactors do have an agitator system, an oxygen delivery system and a foam control system, they provide optimal growth conditions (temperature, pH, substrate, salts, vitamins, oxygen), and the most commonly used ones are of the stirring type.

Main steps in the formation of recombinant DNA in correct sequence are: B: Cutting of DNA at specific location by restriction enzyme. C: Isolation of desired DNA fragment by electrophoresis. D: Amplification of gene of interest using PCR. A: Insertion of recombinant DNA into the host cell.

To create recombinant DNA, both the vector DNA and the source (foreign) DNA are typically cut using the same restriction enzyme so that the sticky/complementary ends generated can anneal together — hence Hind II (or the same enzyme) is used to cut both.

In the pBR322 vector, the tetracycline resistance ($tet^R$) gene contains unique restriction sites for Bam HI and Sal I, which are commonly used for inserting foreign DNA at this locus.

EcoRI is a restriction endonuclease that recognises the specific palindromic sequence GAATTC (C) and cuts the DNA strand between the G and A bases (D), producing sticky/cohesive ends (B), not blunt ends (A is incorrect). It is an endonuclease, not an exonuclease (E is incorrect). So the correct statements are B, C and D only.

RNA interference operates in all eukaryotic organisms as a defense mechanism against invading genetic elements (viruses, transposons), so Statement I is true. RNAi involves the binding of a complementary RNA molecule to a specific mRNA, silencing it and preventing its translation, so Statement II is also true.

Bioreactor is used for production of large quantities of products (a-ii); Electrophoresis is used for separation of DNA fragments (b-i); PCR is used for amplification of nucleic acids (c-iv); ELISA is used for detection of pathogen based on antigen-antibody reaction (d-iii).

Gel electrophoresis separates DNA fragments by size, using an electric field to move negatively charged DNA fragments through a gel matrix, with smaller fragments moving faster/farther than larger ones.

In this context, antibiotics are used to keep the culture medium free from unwanted/contaminating bacteria, while antibiotic resistance genes act as selectable markers to differentiate transformed from non-transformed cells.

Hind II was the first restriction endonuclease discovered, found to always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs (a palindromic sequence).

Restriction enzyme digestion involves incubating purified DNA molecules with the appropriate restriction enzymes under optimum reaction conditions (temperature, buffer, etc.) to cut the DNA at specific sites.

Spooling refers to the process of collecting precipitated/isolated DNA, which appears as fine threads, typically by winding it around a glass rod after precipitation with chilled ethanol.

The thermostable DNA polymerase (Taq polymerase) used in PCR was isolated from the thermophilic bacterium Thermus aquaticus, which allows it to withstand the repeated high-temperature denaturation steps of the PCR cycle.

The $BamHI$ restriction site lies within the tetracycline resistance ($tet^R$) gene of pBR322, so ligating foreign DNA at this site disrupts (inactivates) the $tet^R$ gene, resulting in loss of tetracycline resistance.

Agrobacterium tumefaciens is used primarily as a vector for delivering genes of interest into plant (eukaryotic) host cells, not prokaryotic cells — so the statement that it is used for both prokaryotic and eukaryotic hosts is incorrect.

Kanamycin is an antibiotic used as a selection agent, related to antibiotic resistance (A-IV); ClaI is a restriction enzyme with a specific restriction site (B-III); Disarmed retroviruses are used to deliver genes into animal cells (C-I); the Kanamycin resistance gene ($Kan^R$) acts as a selectable marker (D-II).

Restriction enzymes (restriction endonucleases) cut DNA at specific recognition sequences, acting as 'molecular scissors', while DNA ligase joins DNA fragments together.

Each cycle of PCR has three steps in a fixed order: denaturation, annealing and extension.
Step A is denaturation: the double-stranded DNA is heated to a high temperature (about $94^\circ C$) so that the two strands separate. It is not annealing, and it does not occur at about $50^\circ C$.
Step B is annealing: two sets of primers bind to the complementary regions of the separated strands at a lower temperature. It is not denaturation.
Step C is extension: the thermostable DNA polymerase (Taq polymerase from $Thermus\ aquaticus$) extends the primers using the nucleotides provided, copying the template.
Hence the correct identification is C – extension in the presence of heat stable DNA polymerase.

A plasmid is a small circular DNA that replicates independently in bacteria.
It can be cut with a restriction enzyme and a piece of foreign (e.g. eukaryotic) DNA can be joined into it with DNA ligase, forming recombinant DNA. When put back into a bacterium, the plasmid replicates and copies the foreign gene too.
Antibiotic resistance genes are useful as selectable markers, but the basic reason a plasmid is used as a vector is its ability to carry joined foreign DNA and replicate.
So, as per the answer key, plasmid is used as a vector because it is a circular DNA that can be joined to eukaryotic DNA.

Non-recombinant bacteria have an intact beta-galactosidase gene, so they act on the chromogenic substrate and give blue colonies.
In recombinants the insert inactivates this gene (insertional inactivation), so their colonies stay white.

Polyethylene glycol (PEG) alters the cell membrane; it makes plant protoplasts take up DNA directly from the surrounding medium, and it also induces the fusion of protoplasts.
So PEG-mediated uptake is a chemical method of direct, vector-less gene transfer.
Other vector-less methods are micro-injection, electroporation and biolistics (gene gun).
It has no role in energy production from sewage, biodiesel production or seedless fruit production.
Hence the polyethylene glycol method is used for gene transfer without a vector.

Agrobacterium tumefaciens infects wounds of many dicotyledonous plants and causes crown gall disease, a tumour-like growth.
It does this by transferring a piece of DNA called T-DNA from its tumour-inducing (Ti) plasmid into the plant genome. The T-DNA genes make the plant cells produce growth hormones (causing the gall) and opines, which the bacterium uses as food.
Because this natural gene transfer is the basis of plant genetic engineering (Agrobacterium is called a natural genetic engineer), the Agrobacterium–plant interaction has been studied in great molecular detail.
Stem nodulation of Sesbania, phosphate-solubilising bacteria and the Anabaena–Azolla symbiosis are important but have been studied much less thoroughly.
So the most thoroughly studied bacteria-plant interaction is gall formation on certain angiosperms by Agrobacterium.