How Scientists Cut and Paste Genes With Molecular Scissors

Biotechnology uses living organisms and their molecules to make useful products, from insulin to pest-resistant crops. Modern biotechnology rests on the ability to cut, join and copy genes, and to grow the cells that carry them without contamination.

This part covers what biotechnology is, restriction enzymes, cloning vectors and selection, and ways of getting genes into cells.

The European Federation of Biotechnology defines biotechnology as the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services; modern biotechnology depends on genetic engineering, which alters genetic material, and on a sterile, contamination-free environment in which only the desired microbes or cells grow.

Genetic engineering:

- Changes the chemistry of genetic material, DNA or RNA
- Introduces that material into host organisms, changing their phenotype
- Creates recombinant DNA by joining DNA from different sources

Sterile ambience:

- Allows only the desired microbe or eukaryotic cell to grow in large quantities
- Essential for manufacturing antibiotics, vaccines and enzymes

Why traditional breeding is limited. Hybridisation brings in many unwanted genes along with the desired one, whereas genetic engineering transfers only the chosen gene.

An everyday example. Insulin used by people with diabetes in India is made by genetically engineered microbes grown in sterile fermenters.

The substance. A foreign gene alone cannot multiply in a new host — it must be linked to DNA that has an origin of replication there.

Genetic modification involves identifying DNA with the desirable gene, introducing it into a host, and maintaining it so it passes to the progeny; restriction enzymes cut DNA at specific palindromic recognition sequences, often leaving single-stranded overhangs called sticky ends that let DNA pieces join.

Three basic steps:

- Identify DNA carrying the desirable gene
- Introduce that DNA into the host
- Maintain the introduced DNA in the host and pass it to the progeny

Restriction enzymes:

- They are nucleases; restriction endonucleases cut within DNA, while exonucleases remove nucleotides from the ends
- Each recognises a palindromic sequence, which reads the same on both strands in the 5' to 3' direction, such as 5'-GAATTC-3' paired with 3'-CTTAAG-5'
- Cutting slightly away from the centre of the palindrome, between the same two bases on each strand, leaves sticky ends
- Sticky ends pair with complementary ends cut by the same enzyme, and DNA ligase seals them

Naming EcoRI:

- E — genus Escherichia
- co — species coli
- R — strain RY13
- I — the order in which the enzyme was isolated from that strain

An everyday example. The word MALAYALAM reads the same forwards and backwards — DNA palindromes read the same on both strands in the same direction.

The substance. The same enzyme must cut both the vector and the foreign DNA, so that their sticky ends match.

A cloning vector needs an origin of replication to multiply, a selectable marker to identify transformed cells, and cloning sites where foreign DNA can be inserted; recombinants are picked out by insertional inactivation, in which inserted DNA disrupts a marker gene, so recombinant colonies stay white while non-recombinants turn blue.

Origin of replication (ori). The sequence where replication starts; it also controls the copy number of the linked DNA.

Selectable marker. Helps eliminate non-transformants; genes for resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin are commonly used.

Cloning sites. A vector should have very few, preferably single, recognition sites for the restriction enzyme used, so that it is cut only once.

Example — pBR322. It carries ori, genes for **ampicillin resistance (amp${}^R$) and tetracycline resistance (tet${}^R$)**, and several restriction sites; inserting DNA at the BamHI site inside tet${}^R$ makes recombinants lose tetracycline resistance.

Blue-white screening:

- Foreign DNA is inserted into the gene for **$\beta$-galactosidase, inactivating it
- On a medium with a chromogenic substrate, colonies with a working enzyme turn blue
- Recombinant colonies, with the gene disrupted, stay white

An everyday example. A guard checking passes at a school gate separates authorised visitors from everyone else, just as a selectable marker separates transformed cells from untransformed ones.

The substance. Blue-white screening needs only one plate**, unlike replica plating on two different antibiotics.

Agrobacterium tumefaciens naturally transfers part of its Ti plasmid into plant cells, so a disarmed Ti plasmid serves as a plant vector, and disarmed retroviruses carry genes into animal cells; bacterial cells are made competent with divalent calcium ions and heat shock, while other cells receive DNA by microinjection or biolistics.

Plant vectors:

- Agrobacterium tumefaciens, a plant pathogen, transfers T-DNA from its Ti plasmid, turning normal plant cells into tumour cells
- A disarmed Ti plasmid no longer causes tumours but still delivers useful genes

Animal vectors:

- Retroviruses can turn normal animal cells cancerous
- Disarmed retroviruses deliver desirable genes safely

Getting DNA into host cells:

- Calcium treatment and heat shock — bacteria treated with **Ca${}^{2+}$ are incubated on ice with recombinant DNA, briefly heated to about 42 °C and returned to ice
- Microinjection — recombinant DNA injected directly into the nucleus of an animal cell
- Biolistics or the gene gun — plant cells bombarded with gold or tungsten micro-particles coated with DNA
- Disarmed pathogen vectors — infect cells and transfer recombinant DNA

An everyday example. A courier who delivers parcels without keeping them is like a disarmed vector, which delivers genes without causing disease.

The substance. Pathogens make excellent vectors because they already know how to enter cells** — scientists only remove the disease-causing genes.