Making a Recombinant DNA: Quick Revision

A brief note on Making a Recombinant DNA. Read it before a test. Try each question at the end before you open its answer.

The order of the steps

• Isolate the DNA. Cut it and the vector with the same enzyme. Separate the fragments and elute the wanted one.
• Amplify the gene by PCR. Ligate it into the vector. Put the recombinant DNA into a host.
• Select the transformants (marker), and then the recombinants (insertional inactivation).

Isolation

• Wall: lysozyme for bacteria, cellulase for plants, chitinase for fungi. Animal cells have no wall.
• Cleaning: ribonuclease removes RNA, protease removes proteins.
• Ethanol: chilled ethanol makes the DNA come out as fine threads.

Gel electrophoresis

• Gel: agarose. Charge: DNA is negative, so it moves to the anode.
• Size: the smaller the fragment, the farther it moves.
• Seeing it: ethidium bromide and UV light give orange bands. The wanted band is cut out and eluted.

PCR

• Needs: template, two primers, nucleotides, Taq polymerase (Thermus aquaticus).
• Cycle: denaturation, annealing, extension. The numbers double: $2^n$ after $n$ cycles.
• About one billion: after 30 cycles.

Host and selection

• Bacteria: calcium, ice with the DNA, brief heat shock at 42 degrees Celsius, ice.
• Animal cell: microinjection into the nucleus. Plant cell: gene gun with gold or tungsten particles.
• Disarmed pathogens: Agrobacterium tumefaciens for plants, retroviruses for animal cells.
• BamHI in tetR: recombinants grow on ampicillin and not on tetracycline.
• lacZ: blue colonies have no insert. White colonies are the recombinants.

Practice with new numbers

A program to try

The program shows how fast PCR makes copies.

Mistakes to avoid

• Sending DNA to the cathode. DNA is negative and moves to the anode.
• Saying that ampicillin finds the recombinants. It finds the cells with a plasmid. The broken marker finds the recombinants.
• Calling the blue colonies recombinants. The white ones are.
• Using the wrong wall enzyme. Match the enzyme to the cell.

Check yourself

1. Which enzyme opens a fungal cell?

2. What are the three steps of a PCR cycle, in order?

3. In which direction do the smallest fragments of DNA run in a gel?

4. After 10 cycles of PCR from one molecule, how many molecules are there?

Sources

• NCERT (2023). Biology, Textbook for Class XII, chapter 9.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.