Biotechnology: Principles and Processes, on the exam screen.

Bacillus thuringiensis (Bt) cotton is a classic genetically modified organism (P-II); the thermostable DNA polymerase (Taq polymerase used in polymerase chain reaction (PCR)) is isolated from the bacterium Thermus aquaticus (Q-III); the Ti plasmid is derived from Agrobacterium tumefaciens (R-I); and plasmid constructed by Bolivar and Rodriguez, number 322 (pBR322) is a cloning vector of Escherichia coli (S-IV). This gives P-II, Q-III, R-I, S-IV.

Each PCR cycle proceeds as denaturation (heat separates the two DNA strands), then annealing (primers bind to the single-stranded templates), then extension (DNA polymerase synthesises the new strands). Hence the correct order is Denaturation, then Annealing, then Extension.

Somatic hybridisation starts by isolating single cells from two different plant varieties (Statement IV), digesting their cell walls with enzymes (Statement I) to release naked protoplasts (Statement II), fusing the protoplasts to form a hybrid protoplast (Statement III), and finally growing it to regenerate a new plant (Statement V). Hence the correct sequence is IV, I, II, III, V.

Restriction endonucleases cut DNA a little away from the centre of the palindrome (between the same two bases on the two strands), not exactly at the centre, so statement III is untrue. They cut within the DNA; removing nucleotides from the ends is the job of exonucleases, so statement IV is also untrue. Statements I, II and V correctly describe them. Hence III and IV only are not true.

Restriction enzymes act as molecular scissors that cut DNA (I correct), and the resulting fragments separate by size on an agarose gel during electrophoresis, with smaller fragments migrating farther (II correct). Statement III is wrong because DNA cannot be seen without staining, and Statement IV is wrong because ethidium-bromide-stained DNA is visualised under ultraviolet (UV) light, not in visible light. Hence I and II.

In plasmid constructed by Bolivar and Rodriguez, number 322 (pBR322) the BamHI recognition site lies within the tetracycline-resistance gene (tetR). Ligating foreign DNA at this site disrupts the tetR gene, so the recombinant plasmid loses tetracycline resistance (insertional inactivation) while retaining ampicillin resistance. Hence only tetracycline resistance is lost.

Polymerase chain reaction (PCR) cycles through three steps. Denaturation at about 94 °C separates the two DNA strands. ANNEALING at a lower temperature, typically 50-60 °C, lets the two short oligonucleotide primers base-pair with their complementary sequences on the separated strands. Extension then follows, with Taq polymerase building new strands from those primers. NCERT Class 12, Biotechnology: Principles and Processes.

Both statements are true and together explain why plasmids make such useful cloning vectors. They are small circular DNA molecules that sit OUTSIDE the bacterial chromosome, and because each carries its own origin of replication (ori) it can replicate independently of the host chromosome. Any foreign DNA spliced into a plasmid is therefore multiplied along with it. NCERT Class 12, Biotechnology: Principles and Processes.

DNA is locked inside cells behind a wall and membrane, so the first step in any recombinant DNA procedure is to break the cell open. The enzyme chosen must match the wall chemistry: lysozyme digests the peptidoglycan of bacterial walls, cellulase digests the cellulose of plant walls, and chitinase is used for fungi. The need to disrupt cells is therefore exactly why these particular enzymes are chosen, so R explains A. NCERT Class 12, Biotechnology: Principles and Processes.

Transformation is the process by which recombinant DNA is taken up by a host bacterium (P-II). The cloning site is the recognition sequence where a restriction enzyme cuts the vector so foreign DNA can be inserted (Q-I). Selection uses an antibiotic resistance marker to identify which cells took up the vector (R-IV). The ori is the origin of replication, the sequence that allows the vector to be copied inside the host (S-III). NCERT Class 12, Biotechnology: Principles and Processes.

Bacillus thuringiensis supplies Cry proteins; Thermus aquaticus supplies thermostable DNA polymerase; Agrobacterium tumefaciens is a plant cloning vector; and Salmonella typhimurium was used in the first recombinant-DNA construction. Thus P-IV, Q-III, R-I, S-II. Chapter: Biotechnology: Principles and Processes.

DNA ligase joins DNA fragments by sealing breaks in the sugar-phosphate backbone. It forms phosphodiester bonds between adjacent 3′-hydroxyl and 5′-phosphate ends.

Separated DNA fragments in gel electrophoresis are visualized using ethidium bromide, which intercalates with DNA and fluoresces under ultraviolet (UV) radiation. Chapter: Biotechnology: Principles and Processes.

Each restriction enzyme recognizes and cuts DNA at specific palindromic sequences, not by inspecting the length of a DNA sequence. Chapter: Biotechnology: Principles and Processes.

Restriction enzyme from Escherichia coli RY 13 (EcoRI) recognizes the palindromic sequence 5' - GAATTC - 3' and its complementary strand 3' - CTTAAG - 5'. Chapter: Biotechnology: Principles and Processes.

The origin of replication (Ori site) controls the copy number of the linked DNA in a vector. This site is essential for the initiation of DNA replication and determines how many copies of the plasmid are made within the host cell. Chapter: Biotechnology: Principles and Processes.