Q.List the sequential steps in the process of recombinant DNA technology, starting from isolation of the desired DNA fragment and ending with obtaining the desired product.

Chitinase digests chitin, the main structural polysaccharide of fungal cell walls, allowing release of the protoplast.

To obtain DNA/RNA or make a protoplast for genetic engineering, the cell wall must first be removed enzymatically. Bacterial cell walls are digested by lysozyme, plant cell walls by cellulase and pectinase, and fungal cell walls (made mainly of chitin) are digested by chitinase, releasing the protoplast for further manipulation such as transformation.

To isolate DNA from a fungal cell, you need an enzyme that breaks down the tough, chitin-rich cell wall — and that enzyme is chitinase.

Treating a cell extract with the enzyme ribonuclease selectively degrades RNA while leaving DNA intact, useful during recombinant DNA/plasmid isolation.

During the isolation of genetic material for biotechnology (e.g., preparing pure DNA/plasmid), the extract typically contains other macromolecules like RNA and protein along with the desired DNA. RNA is specifically removed by treating the preparation with the enzyme ribonuclease (RNase), which hydrolyses RNA molecules, while proteins are removed separately using protease.

The assertion about using specific enzymes to degrade cell walls for DNA isolation is true, but the reason is false because fungal cell walls are degraded by chitinase (not cellulase, which acts on plant cellulose). The correct option is (C).

DNA isolation needs the cell wall removed first; a fungal wall (chitin) is digested with chitinase.

To isolate DNA, the cell must first be opened by digesting its wall with an appropriate enzyme. The enzyme chosen depends on the cell wall's composition: lysozyme for bacteria (peptidoglycan), cellulase for plant cells (cellulose), and chitinase for fungi, whose cell walls are made of chitin. DNase (option c) would degrade the very DNA we want to isolate, so it is never used for this step.

After alcohol precipitation, DNA appears as fine threads; it is pelleted and separated from the rest of the solution by centrifugation.

In recombinant DNA technology, once cells/tissue are lysed with enzymes (lysozyme for bacteria, cellulase for plant cells, chitinase for fungi) to release DNA along with other macromolecules (RNA, proteins, polysaccharides, lipids), these contaminants are first removed by treatment with RNase, protease and other steps. The purified DNA is then made to precipitate out of the chilled solution by adding chilled ethanol, where it becomes visible as fine collapsed threads. To separate this precipitated DNA (and other biomolecules) from the rest of the solution, the mixture is subjected to centrifugation — spinning at high speed causes the denser precipitated material to pellet at the bottom of the tube, from where it can be separated from the supernatant.

Lysozyme digests bacterial cell walls, cellulase digests plant cell walls, and chitinase digests fungal cell walls — all used to prepare protoplasts.

To isolate genetic material or produce protoplasts for genetic engineering, the cell wall of the host organism must first be removed using an appropriate wall-degrading enzyme, matched to the composition of that organism's cell wall:

• Bacteria — cell walls (made mainly of peptidoglycan) are digested using the enzyme lysozyme.
• Plant cells — cell walls (made of cellulose) are digested using the enzyme cellulase.
• Fungus — cell walls (made of chitin) are digested using the enzyme chitinase.

So the correct matching is: i) Bacteria → c) Lysozyme; ii) Plant cells → a) Cellulase; iii) Fungus → b) Chitinase — i.e., i(c), ii(a), iii(b), matching option (b).

To isolate DNA/proteins from bacteria, the cell wall is broken using the enzyme lysozyme, which digests the peptidoglycan layer.

Before DNA can be extracted and manipulated in genetic engineering, the source cell must be broken open (lysed) to release its cellular contents.

• Bacterial cells are treated with lysozyme, an enzyme that specifically degrades the peptidoglycan (murein) layer of the bacterial cell wall, causing the cell to burst (lyse) and release its contents including DNA, RNA, and proteins.

• (In contrast, plant cells are broken using cellulase, and fungal cells using chitinase, since their cell walls are made of different polymers.)

Genetic engineering always begins with isolating the DNA/gene of interest from the organism that carries it.

The process of genetic engineering follows a defined sequence of steps: identification and isolation of the desired genetic material (DNA) carrying the gene of interest from the source organism; cutting this DNA and a chosen vector using restriction enzymes; joining (ligating) the gene of interest into the vector to form recombinant DNA; introducing this recombinant DNA into a suitable host cell (transformation); and finally selecting the transformed host cells and allowing them to express and multiply the desired gene product. Isolation of genetic material (DNA) is therefore always the essential first step, since without the target DNA in hand, none of the subsequent cutting, ligation or transfer steps can be carried out.