Why DNA Cannot Just Walk In

Having built a recombinant vector, the next hurdle is getting it inside a living cell. This is harder than it sounds. DNA is a hydrophilic molecule — it is comfortable in water but repelled by the oily interior of a membrane. Because of this, DNA cannot pass through cell membranes on its own.

The cell membrane, in other words, is a barrier the DNA cannot cross unaided. So before a bacterium can take up a plasmid, we have to change the bacterium itself, making it willing and able to accept foreign DNA. A cell prepared in this way is described as competent.

Making Bacteria Competent

To make bacterial cells competent, they are treated with a specific concentration of a divalent cation, most commonly calcium (Ca2+). This treatment increases the efficiency with which DNA can enter the bacterium through the pores in its cell wall. The cations, in effect, loosen the way and make the wall more permeable to the incoming plasmid.

With the cells now competent, the recombinant DNA is forced in by a short temperature trick. The competent cells are incubated with the recombinant DNA on ice, then given a brief heat shock at 42 degrees Celsius, and immediately put back on ice. This sudden shift encourages the bacteria to take up the recombinant DNA from their surroundings.

Micro-injection and the Gene Gun

The calcium-and-heat-shock method is not the only route in. For animal cells, one option is micro-injection: the recombinant DNA is injected directly into the nucleus of the cell with a fine needle, bypassing the membrane barrier altogether.

For plant cells, there is biolistics, also called the gene gun. Here the cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. The tiny particles punch through the cell wall and membrane, carrying the DNA in with them. The name captures the idea neatly — a biological ballistics, firing DNA-loaded pellets into the target cells.

Letting a Disarmed Pathogen Do the Work

The final method turns back to the vectors we met earlier: disarmed pathogens. Instead of physically forcing DNA across the membrane, we let a harmless, engineered pathogen do what pathogens do best. When a disarmed-pathogen vector is allowed to infect the cell, it naturally transfers the recombinant DNA into the host as part of its ordinary infection route.

So across all these approaches the goal is identical — get the recombinant DNA past the membrane — while the means differ: chemically prime the cell (competence plus heat shock), inject it, shoot it in, or let a tamed pathogen ferry it in.

Quick Recap

  • DNA is hydrophilic, so it cannot cross cell membranes on its own.
  • Bacteria are made competent by treatment with a divalent cation such as calcium (Ca2+), which improves DNA entry through pores in the cell wall.
  • Recombinant DNA is then forced in by incubating the competent cells with it on ice, giving a brief heat shock at 42 degrees Celsius, and returning them to ice.
  • Micro-injection delivers recombinant DNA straight into the nucleus of an animal cell.
  • Biolistics (gene gun) bombards plant cells with high-velocity micro-particles of gold or tungsten coated with DNA.
  • Disarmed-pathogen vectors infect the cell and transfer the recombinant DNA into the host.

Solved Examples — Section 10

Q1. Why is DNA unable to pass through a cell membrane on its own?

Answer: Because DNA is a hydrophilic molecule, and so it cannot pass through the hydrophobic cell membrane unaided.

Q2. How are bacterial cells made competent to take up DNA?

Answer: They are treated with a specific concentration of a divalent cation such as calcium (Ca2+), which increases the efficiency with which DNA enters through pores in the cell wall.

Q3. Describe the heat-shock step used to force recombinant DNA into competent cells.

Answer: The competent cells are incubated with the recombinant DNA on ice, given a brief heat shock at 42 degrees Celsius, and then placed back on ice, which enables uptake of the DNA.

Q4. What is micro-injection?

Answer: A method in which recombinant DNA is injected directly into the nucleus of an animal cell.

Q5. What are the micro-particles coated with in biolistics, and what is this method also called?

Answer: They are micro-particles of gold or tungsten coated with DNA; the method is also called the gene gun.

Q6. How does a disarmed-pathogen vector introduce recombinant DNA into a cell?

Answer: It is allowed to infect the cell, and in doing so it transfers the recombinant DNA into the host.