How Scientists Cut and Paste Genes Using Molecular Scissors

Human insulin today is made by bacteria. That is possible because scientists can cut a gene out of one organism's DNA, paste it into a carrier DNA, and slip it into a host cell that copies and uses it.

This lesson covers DNA isolation, making recombinant DNA, vectors and gene transfer, and selecting, amplifying and producing the products of recombinant cells.

Biotechnology uses living organisms, cells or their parts to make useful products and processes, and DNA is isolated by breaking open cells, removing other molecules with enzymes and precipitating pure DNA with chilled ethanol, after which the gene of interest is cut out and separated by gel electrophoresis.

Isolating genomic DNA:

- Break open cells with enzymes — lysozyme for bacteria, cellulase for plant cells and chitinase for fungi
- Remove RNA with ribonuclease and proteins with protease
- Add chilled ethanol, so purified DNA precipitates as fine threads that can be spooled out

Isolating the gene of interest:

- Cut the DNA with restriction enzymes
- Separate the fragments by agarose gel electrophoresis — negatively charged DNA moves towards the positive electrode, and smaller fragments travel farther
- Stain with ethidium bromide and view the orange bands under UV light
- Cut out the band with the desired gene and recover it — elution

An everyday example. Science fair demonstrations that pull white DNA threads out of mashed banana or onion use the same steps — breaking cells, removing proteins and adding cold alcohol.

The substance. Gel electrophoresis sorts DNA by size, not by sequence — two different genes of the same length would stop at the same position.

Recombinant DNA is made by cutting both the source DNA and the vector with the same restriction enzyme, so that their sticky ends match, and joining the pieces with DNA ligase.

Restriction enzymes:

- Restriction endonucleases cut DNA at specific sites within the molecule
- They recognise palindromic sequences, which read the same on both strands in the 5' to 3' direction
- Many cut the two strands slightly off-centre, leaving single-stranded sticky ends that pair with complementary ends

Naming, using EcoRI:

- E — genus Escherichia; co — species coli
- R — the strain, RY 13
- I — the order in which the enzyme was isolated from that strain

Worked example. EcoRI recognises

$$5^{\prime }\text{-GAATTC-}{3}^{\prime }\text{paired with}{3}^{\prime }\text{-CTTAAG-}{5}^{\prime }$$

and cuts between G and A on each strand, leaving AATT overhangs as sticky ends.

Joining. DNA ligase seals the sugar-phosphate backbone between the vector and the inserted gene, forming a stable recombinant DNA molecule.

An everyday example. Two pieces of cloth cut with the same zigzag scissors fit together neatly before stitching, just as matching sticky ends pair before ligase seals them.

The substance. The same enzyme must cut both DNAs — different restriction enzymes usually leave ends that cannot pair.

Cloning vectors are DNA molecules such as plasmids that carry a foreign gene into a host and replicate there, and genes are transferred into hosts by making cells competent, by microinjection, by gene guns or by disarmed pathogens.

Features of a cloning vector:

- Origin of replication (ori) — where replication starts; it controls the copy number
- Selectable marker — such as a gene for resistance to ampicillin or tetracycline, to identify transformed cells
- Cloning sites — ideally a single recognition site for the chosen restriction enzyme

Examples of vectors:

- Plasmid pBR322 — carries resistance genes for ampicillin and tetracycline, with sites for several restriction enzymes
- Ti plasmid of Agrobacterium tumefaciens — normally causes tumours in dicot plants, but a disarmed version carries useful genes into plants
- Disarmed retroviruses — deliver genes into animal cells

Methods of gene transfer:

- Competent cells — bacteria treated with calcium ions and given a heat shock, moving from ice to 42 °C and back, take up DNA
- Microinjection — DNA is injected directly into the nucleus of an animal cell
- Biolistics (gene gun) — plant cells are bombarded with tiny gold or tungsten particles coated with DNA

An everyday example. Developing insect-resistant cotton relies on these methods to place a bacterial gene inside cotton cells.

The substance. A vector should have only one site for the chosen restriction enzyme — several sites would chop the vector into pieces.

Recombinants are identified by insertional inactivation of a marker gene, genes are amplified by the polymerase chain reaction (PCR), and the product is made on a large scale in bioreactors and purified by downstream processing.

Selecting recombinants:

- Antibiotic markers — inserting a gene into the tetracycline resistance gene of pBR322 means recombinants grow on ampicillin but not on tetracycline
- Blue-white selection — the lacZ gene makes beta-galactosidase, which turns a chromogenic substrate blue; inserting a foreign gene inactivates it, so recombinant colonies are white and non-recombinants blue

PCR — amplifying a gene:

- Denaturation — heating separates the two DNA strands
- Annealing — short primers bind to the ends of the target region
- Extension — heat-stable Taq polymerase, from Thermus aquaticus, builds new strands
- Each cycle doubles the DNA, so $n$ cycles give $2^n$ copies from one molecule

Worked example. After 20 cycles, one DNA molecule gives

$$2^{20}=1048576\approx 1\text{ million copies}$$

Bioreactors and downstream processing:

- Bioreactors, often stirred-tank types, give recombinant cells the right temperature, pH, oxygen and nutrients
- Downstream processing separates and purifies the product and formulates it with preservatives

An everyday example. Insulin sold at Indian pharmacies is made by growing recombinant cells in bioreactors and purifying the protein through downstream processing.

The substance. Blue-white selection needs only one plate — unlike testing on two antibiotics, recombinants are picked out directly by colour.