Biology · Biotechnology: Principles and Processes · NEET
Because DNA is a hydrophilic (water-loving) molecule and carries a negative charge, so it cannot pass through the cell membrane, which has a hydrophobic (water-hating) inner layer. NCERT states this directly: 'Since DNA is a hydrophilic molecule, it cannot pass through cell membranes.' This is exactly why the cell must first be made competent before DNA can enter.
A competent cell is a bacterial cell that has been treated so it can now take up DNA from its surroundings. Normal cells cannot do this. To make E. coli competent, the cells are treated with a specific concentration of a divalent cation such as calcium (CaCl2). This increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
Calcium (Ca2+) is a divalent cation, meaning it carries two positive charges. DNA is negatively charged, so the Ca2+ ions neutralise the negative charge of both the DNA and the cell surface. This reduces repulsion and lets DNA cling near the pores in the cell wall, so it can slip in during the heat shock. For NEET, remember the exact word: divalent cation such as calcium.
The steps are: incubate competent cells with recombinant DNA on ice, then place them briefly at 42 C (heat shock), then put them back on ice. The sudden temperature change creates a pressure difference that drives the DNA through the cell wall pores. Cold, hot, cold is the exact NCERT sequence and a common one-mark trap.
For bacteria, yes. Transformation is the procedure through which a piece of DNA is introduced into a host bacterium. When that DNA is recombinant DNA carrying, say, an ampicillin-resistance gene, the host cell becomes transformed into an ampicillin-resistant cell. So transformation is one specific method of insertion, used for bacterial hosts.
NCERT gives three other methods besides transformation. Microinjection: recombinant DNA is directly injected into the nucleus of an animal cell. Biolistics or gene gun: plant cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. Disarmed pathogen vectors: a harmless pathogen (like a disarmed Ti plasmid of Agrobacterium) infects the cell and transfers the recombinant DNA.
Main steps in the formation of Recombinant DNA are given below. Arrange these steps in a correct sequence. A. Insertion of recombinant DNA into the host cell. B. Cutting of DNA at specific location by restriction enzyme. C. Isolation of desired DNA fragment. D. Amplification of gene of interest using PCR.
Match List-I with List-II: A. Transformation B. Cloning site C. Selection D. Ori | I. Restriction enzyme II. Transfer DNA to host bacteria III. Replication IV. Antibiotic
Try the real previous-year questions from this chapter — each with the answer and a full solution.
A bacterial cell treated with a divalent cation (like calcium) so it can now take up DNA from its surroundings, which normal cells cannot do.
42 C, given briefly, with the cells kept on ice before and after the shock.
Microinjection, in which recombinant DNA is directly injected into the nucleus of an animal cell.
For plant cells: they are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA.
Because the DNA must first be cut, joined into a vector and amplified; only the finished recombinant DNA is then inserted into the host so it can multiply and later express the product.
