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Cutting and joining DNA gets most of the attention in the biotechnology chapter. But a recombinant plasmid sitting in a test tube does nothing. It has to get inside a living cell, and a cell membrane is built to keep it out. This post explains why, sets out the four methods NCERT names, looks at the experiments where two of them were first shown to work, and ends with a worked selection problem.
Where is this in the syllabus?
The NEET (UG) 2026 syllabus lists "Principles and process of Biotechnology: Genetic engineering (Recombinant DNA technology)" in Unit 9. The methods are in sections 9.2.3 and 9.3.4 of NCERT Class 12 Biology Chapter 9, Biotechnology: Principles and Processes (2026-27 reprint), with disarmed vectors in its section on cloning vectors. Check your own year's syllabus.
Why can DNA not just enter a cell?
NCERT's answer is one line: "Since DNA is a hydrophilic molecule, it cannot pass through cell membranes." The membrane's interior is lipid, and a large, charged, water-loving molecule cannot cross it. Every method below is a way of forcing the issue.
Method 1: competent bacteria and heat shock
Treat the bacteria with a specific concentration of a divalent cation, such as calcium. NCERT says this increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Cells treated this way are called competent.
Incubate the cells with the recombinant DNA on ice.
Move them briefly to 42 °C (the heat shock).
Put them back on ice.
The bacteria then take up the recombinant DNA.
Method 2: microinjection into animal cells
In microinjection, recombinant DNA is injected directly into the nucleus of an animal cell. An early test of this was reported by Gordon and colleagues (1980). They microinjected a recombinant plasmid, built on pBR322, into the pronuclei of fertilised mouse eggs and let the embryos develop to term. In one series, 2 of 78 newborn mice, about 2.6 per cent, carried DNA matching the injected plasmid, though rearranged. The authors concluded that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos.
Method 3: biolistics or the gene gun, for plants
Plant cells have a tough wall. NCERT's method "suitable for plants" bombards cells with high-velocity micro-particles of gold or tungsten coated with DNA. Klein and colleagues (1987) reported the idea in *Nature*: accelerated tungsten particles pierced cell walls and membranes and entered intact onion epidermal cells without killing them, and the RNA or DNA they carried was then expressed. They hoped it would get around limits of existing methods, including the host range of *Agrobacterium tumefaciens*.
Method 4: disarmed pathogens
Some pathogens already deliver DNA for a living. NCERT notes that *Agrobacterium tumifaciens* transfers a piece of DNA called T-DNA into dicot plant cells, and retroviruses can transform animal cells. The Ti plasmid has been modified into a vector that is no longer pathogenic but still delivers genes into many plants, and retroviruses have been disarmed to deliver genes into animal cells.
Worked example: did it work? (sample numbers)
NCERT explains how you find the cells that took up the DNA. If the recombinant plasmid carries an ampicillin resistance gene and is put into *E. coli*, then on agar containing ampicillin "only transformants will grow".
Suppose (these are made-up classroom numbers) you heat-shock competent *E. coli* with 0.1 ng of such a plasmid, add broth to make 1 mL, and spread 100 µL on an ampicillin plate. Next day there are 150 colonies.
100 µL is one-tenth of the 1 mL, so the whole tube held 150 × 10 = 1500 transformants.
0.1 ng is 0.0001 µg.
Transformants per µg = 1500 ÷ 0.0001 = 1.5 × 10⁷ per µg of DNA.
Labs use this "transformation efficiency" to compare batches of competent cells. It is beyond the NCERT text, but the logic, selection on an antibiotic, is straight from it. For how pBR322 tells recombinants from plain plasmids, see our post on biotechnology principles for NEET.
Which mistakes cost the most marks?
Microinjecting into the cytoplasm. NCERT says directly into the nucleus.
Using the gene gun for bacteria. NCERT names it as the method suitable for plants.
Heat shock "at 42 K". It is 42 °C, between two spells on ice.
Thinking heat alone makes cells competent. Competence comes from the divalent cation treatment first.
Forgetting the vector is disarmed. NCERT says the modified Ti plasmid is no longer pathogenic to plants.
A practice routine
One-line cards: host, method and one detail for each of the four methods. Test yourself, then check against NCERT sections 9.2.3 and 9.3.4.
Sequence drill: write the heat-shock steps in order from memory.
Two transformation sums with your own sample numbers, forwards and backwards.
Explain aloud why DNA cannot cross a membrane, in one sentence.
When a step in a protocol is hard to picture, the Fermi Simulation Lab has interactive simulations for ideas that are easier to see than read.
Today, rework the sample: 0.5 ng of plasmid, 1 mL recovery, 50 µL plated, 120 colonies. What is the transformation efficiency?
