Recombinant DNA Technology and Restriction Enzymes

Recombinant DNA technology is the joining of DNA from different sources into a single molecule using restriction enzymes to cut DNA at specific sequences and DNA ligase to join the pieces, usually in a plasmid vector that is then introduced into a host cell. In brief: restriction enzymes such as EcoRI cut DNA at specific palindromic sequences (GAATTC for EcoRI) and often leave short single-stranded “sticky ends”. If a gene and a plasmid are cut with the same enzyme, their sticky ends can pair, and DNA ligase seals the join. The recombinant plasmid is taken up by bacteria (transformation), which can then make the protein coded by the gene, as in the production of human insulin.

These methods made genetic engineering possible. This guide defines biotechnology and selective breeding in context, explains restriction enzymes, plasmids and vectors, lists the steps of making a recombinant organism, compares the tools in a table, works through a restriction-digest example with calculated fragment sizes and corrects common mistakes.

What are biotechnology and genetic engineering?

Biotechnology is the use of living organisms or their parts to make products or processes useful to humans. It includes ancient techniques such as brewing, baking, cheese-making and selective breeding of crops and animals, as well as modern methods. Genetic engineering is the direct modification of an organism’s DNA in the laboratory by adding, removing or changing genes. Unlike selective breeding, which works with the existing variation within a species over many generations, genetic engineering can move a specific gene from one species into another in a single step.

What are restriction enzymes?

Restriction enzymes (restriction endonucleases) are enzymes from bacteria that cut DNA at specific recognition sequences. Bacteria use them as a defense against viruses (bacteriophages): they cut the foreign DNA, while the bacterium’s own DNA is protected by chemical modification (methylation) at these sites. Names come from the bacterium: EcoRI is from Escherichia coli strain RY13, HindIII from Haemophilus influenzae, and BamHI from Bacillus amyloliquefaciens.

• Recognition sites are palindromic: the sequence reads the same 5′ to 3′ on both strands. EcoRI recognizes 5′-GAATTC-3′ (the complementary strand also reads 5′-GAATTC-3′).
• Sticky ends: EcoRI cuts between G and A on each strand: 5′-G^AATTC-3′. The cuts are staggered, leaving single-stranded overhangs of AATT, which can pair with any other DNA cut with the same enzyme, through complementary base pairing.
• Blunt ends: some enzymes, such as SmaI (CCC^GGG), cut straight across and leave no overhang. Blunt ends can be joined too, but less efficiently.

A given six-base sequence occurs on average once in every 4⁶ = 4 096 base pairs (assuming that all bases are equally probable and random), so enzymes with six-base recognition sites cut DNA into fragments of a few thousand base pairs on average. Enzymes that recognize four bases cut more often (once every 4⁴ = 256 bp).

What are plasmids and vectors?

A vector is a DNA molecule that is used to carry foreign DNA into a host cell. Bacterial plasmids are the most commonly used: small circular DNA molecules (typically 2 000 to 10 000 bp) that replicate independently of the chromosome. A good plasmid vector has:

• An origin of replication so that it is copied in the host.
• One or more restriction sites (a multiple cloning site) where foreign DNA can be inserted.
• A selectable marker, usually an antibiotic resistance gene (such as ampicillin resistance), so that only the bacteria that have taken up the plasmid survive on a plate with that antibiotic.
• Often a reporter gene such as lacZ (blue-white screening) or green fluorescent protein (GFP), which shows if the insert is present.

Other vectors include bacteriophages and modified viruses (used in gene therapy), yeast artificial chromosomes (for large DNA pieces) and, in plants, the Ti plasmid of the bacterium Agrobacterium tumefaciens.

What are the steps of making recombinant DNA?

1. Isolate the gene: cut it out of the source DNA with a restriction enzyme, or make it from mRNA using reverse transcriptase (producing complementary DNA, or cDNA, which lacks introns), or synthesize it chemically.
2. Cut the plasmid with the same restriction enzyme, so that it has matching sticky ends.
3. Mix and join: the gene and the plasmid pair by their sticky ends, and DNA ligase forms phosphodiester bonds to seal the backbone. This produces the recombinant plasmid.
4. Transformation: the bacteria are made competent to take up DNA, by a treatment with calcium chloride and a heat shock, or by electroporation (a short electric pulse). Only a small fraction of the cells take up the plasmid.
5. Selection: bacteria are grown on agar that contains an antibiotic. Only those with the plasmid, and therefore the resistance gene, form colonies.
6. Screening and growth: colonies that carry the insert are identified, for example by white color in blue-white screening, and grown in large cultures (fermenters), in which they make the protein. The product is purified.

Not all of the plasmids take up the gene: some plasmids simply close up again, and some fragments join together in the wrong way. Treatment of the cut plasmid with a phosphatase enzyme, which prevents its ends from rejoining, and the use of two different enzymes to cut give better results.

The molecular toolkit

Worked example: a restriction digest

A linear DNA molecule of 6 000 base pairs has two EcoRI sites, at positions 1 200 and 3 500 measured from the left end. A circular plasmid of 4 500 bp has one EcoRI site.

1. Fragments from the linear DNA: the two cuts give three fragments: 1 200 bp (0 to 1 200), 3 500 − 1 200 = 2 300 bp (1 200 to 3 500), and 6 000 − 3 500 = 2 500 bp (3 500 to 6 000). Check: 1 200 + 2 300 + 2 500 = 6 000.
2. Number of fragments: a linear molecule cut at n sites gives n + 1 fragments (2 sites give 3 fragments).
3. Plasmid: a circular molecule cut at one site is opened into one linear piece of 4 500 bp. A circular molecule with n sites gives n fragments.
4. Recombinant plasmid: if the 2 300 bp fragment is ligated into the opened plasmid, the new circular plasmid has 4 500 + 2 300 = 6 800 bp. Cutting it with EcoRI would give two fragments: 2 300 and 4 500 bp (if the insert went in with both sites intact).

On a gel (the subject of a later article), the fragments would show as three bands, with the smallest fragment moving the furthest. The fragment size is used as a “fingerprint” to confirm whether the insert is present. As a check on the average frequency, a 6 000 bp DNA with the expected one site per 4 096 bp would contain on average 6 000 ÷ 4 096 ≈ 1.5 sites, so two sites is not unusual.

How was the technology developed?

Werner Arber, Daniel Nathans and Hamilton Smith discovered and characterized restriction enzymes in the 1960s and early 1970s, and shared the 1978 Nobel Prize in Physiology or Medicine. In 1972, Paul Berg made the first recombinant DNA molecules, and in 1973 Stanley Cohen and Herbert Boyer showed that recombinant plasmids could be introduced into bacteria and replicated. In 1975, scientists met at the Asilomar conference in California to discuss safety and set up guidelines. Genentech, founded by Boyer and Robert Swanson in 1976, produced human insulin in bacteria in 1978, and in 1982 the first genetically engineered drug, human insulin (Humulin), was approved.

What are the applications?

Bacteria and yeast are used to make human insulin, growth hormone, clotting factors for hemophilia, hepatitis B vaccine proteins and enzymes for detergents and food processing. Cloned genes are used for research, to study gene function and to make probes and DNA libraries. The same techniques are applied to plants and animals. Safety rules, such as containment in the laboratory and the use of host bacteria that cannot survive outside, were put in place to reduce risks.

Which mistakes are common?

Mistake 1: Saying that restriction enzymes cut at random

They cut only at specific recognition sequences.

Mistake 2: Using different enzymes for the gene and the vector

The ends must be complementary, so the same enzyme (or enzymes that give compatible ends) is used for both.

Mistake 3: Confusing ligase with restriction enzymes

Restriction enzymes cut DNA. Ligase joins it.

Mistake 4: Assuming all bacteria take up the plasmid

Only a small proportion do. The selectable marker identifies them.