Q.Give reasons why :

Concept: Molecular biology principles in recombinant DNA technology

1. DNA cannot pass through the cell membrane The cell membrane is a selectively permeable lipid bilayer that allows only small, non-polar molecules to diffuse freely. DNA is a large, highly negatively charged (due to phosphate groups in its backbone) hydrophilic macromolecule. The hydrophobic core of the membrane repels charged molecules, and the sheer size of DNA (thousands to millions of base pairs) prevents passive diffusion. Cells must therefore be made competent (chemically or by electroporation) to create temporary pores for DNA uptake.
2. Proteases are added during DNA isolation Proteins, especially nucleases and histones, are tightly bound to DNA in cells. Nucleases can degrade the DNA during extraction, while histones and other chromosomal proteins interfere with downstream enzymatic reactions (restriction digestion, ligation). Proteases digest these contaminating proteins into amino acids, ensuring pure, intact DNA suitable for cloning and manipulation.
3. Single cloning site is preferred in a vector …

(a) Why DNA cannot pass into a host cell through the cell membrane

The plasma membrane is a selectively permeable lipid bilayer that controls what enters and exits the cell. Its hydrophobic core allows small, non-polar molecules (like O2, CO2) to diffuse freely, but it acts as a formidable barrier to large, charged molecules.

DNA is a massive, highly negatively charged macromolecule. Consider the obstacles:

1. Size: A typical plasmid used in cloning is 3–10 kilobase pairs, translating to a molecular weight in the millions of Daltons. The phosphodiester backbone forms a long, rigid double helix far too large to slip between the tightly packed phospholipid tails.

2. Charge: Every nucleotide contributes a negatively charged phosphate group. The entire DNA molecule is therefore polyanionic. The hydrophobic interior of the membrane repels charged species — there is no energetically favorable path for a polyanion to traverse the non-polar lipid core.

3. Hydrophilicity: The sugar-phosphate backbone is hydrophilic. Water molecules solvate the DNA in the aqueous environment, and stripping this hydration shell to enter the membrane would require enormous energy.

Because of these barriers, DNA cannot spontaneously diffuse across the cell membrane. Genetic engineers must use artificial methods — electroporation (electric pulses create transient pores), chemical transformation (calcium chloride treatment in bacteria), or microinjection — to force DNA entry.

(b) Why proteases are added during isolation of DNA for genetic engineering

When you lyse cells to extract DNA, you release not just the DNA but the entire cellular contents — including nucleases (DNases and RNases), enzymes that degrade nucleic acids. If left unchecked, these nucleases will chop your precious DNA into fragments, ruining the isolation.

Proteases are added to digest the nucleases themselves, which are proteins. Here's the logic:

1. Nucleases are proteins: DNases and RNases are enzymes with peptide backbones. Proteases (like Proteinase K) cleave peptide bonds, breaking down these enzymes into amino acids or small peptides.

2. Inactivation: Once the nuclease protein is digested, it loses its three-dimensional structure and active site — it can no longer catalyze the hydrolysis of DNA phosphodiester bonds.

3. Preservation of DNA integrity: With nucleases destroyed, the DNA remains intact during the isolation procedure. You can then precipitate, wash, and purify the DNA without degradation.

Additionally, proteases help remove other contaminating proteins (histones, membrane proteins) that might co-precipitate with DNA, yielding a purer final product.

(c) Why a single cloning site is preferred in a vector

A cloning vector (plasmid, phage, cosmid) must carry the foreign DNA insert in a controlled, predictable manner. The cloning site is where you cut the vector with a restriction enzyme and paste in your gene of interest. Having a single (unique) cloning site for a given restriction enzyme is crucial for several reasons:

1. Unambiguous insertion: If the restriction enzyme recognition sequence appears only once in the vector, cutting with that enzyme produces a single, defined break. When you ligate your insert (cut with the same enzyme), it can only go into that one location. There is no ambiguity about where the gene ends up. …