Restriction enzymes

Genetic engineering means taking a gene from one organism and putting it into a different organism. To do this, scientists need a way to cut DNA in exactly the right place and a way to join pieces of DNA together. Two enzymes do these jobs:

• Restriction enzymes — these cut DNA. Think of them as molecular scissors.
• DNA ligase — this joins pieces of DNA together. Think of it as molecular glue.

A useful way to picture the whole process is to imagine cutting a wanted gene out of a long strand of DNA, then sticking it into a small circular loop of bacterial DNA called a plasmid. The plasmid then carries the gene into a bacterium, which can read the gene and make the useful protein (for example, human insulin).

In this subtopic you only need the two 'tool' enzymes and how they work — the cut by restriction enzymes and the join by DNA ligase.

Exam tip. Learn the simple job of each enzyme: restriction enzymes cut, ligase joins. Mixing these up is the most common error in this topic.

A restriction enzyme does not cut DNA just anywhere. It recognises a specific sequence of bases (a short run of A, T, C and G in a particular order) and cuts the DNA only at that sequence. This is why we say it cuts at specific sites.

When many restriction enzymes cut, they do not cut both strands of the DNA straight across. Instead they cut in a staggered way, leaving a short stretch of single-stranded DNA sticking out at each cut end. These overhangs are called sticky ends.

Why are they called 'sticky'? Because the exposed single-stranded bases can pair up with another piece of DNA that has a matching (complementary) sticky end. Remember the base-pairing rule: A pairs with T, and C pairs with G. So a sticky end will only join neatly to another sticky end whose bases are complementary to it.

Here is a simple picture of a restriction enzyme cutting DNA to leave two sticky ends:

Exam tip. Two marks live here: restriction enzymes cut at a specific (base) sequence, and the cut leaves sticky ends. Use the word complementary when you explain why sticky ends join.

This is the idea that examiners reward most, so make sure you understand it.

To insert a gene into a plasmid, scientists cut both the desired gene and the plasmid using the same restriction enzyme.

Why the same enzyme? Because each restriction enzyme cuts at its own specific base sequence and leaves a particular shape of sticky end. If you use the same enzyme on both pieces:

• the sticky end left on the gene and the sticky end left on the plasmid will be complementary to each other;
• so the two pieces have matching overhanging bases that pair up (A–T and C–G);
• which means the gene fits neatly into the gap in the plasmid.

If you used different enzymes, the sticky ends would not match, and the gene could not be slotted into the plasmid. So 'same enzyme → complementary sticky ends → the pieces join' is the chain of reasoning to write down.

Exam tip. A very common question is 'Explain why the same restriction enzyme is used to cut both the gene and the plasmid.' The mark is for complementary / matching sticky ends so the pieces can join.

Once the gene's sticky end has paired up with the plasmid's complementary sticky end, the two pieces are only loosely held together by base pairing. They need to be sealed so they become one continuous, joined-up molecule.

This is the job of DNA ligase. Ligase is an enzyme that joins pieces of DNA together — it 'glues' the gene firmly into the plasmid by sealing the backbone of the DNA.

The result is a single plasmid that now contains the inserted gene. Because this DNA is made by combining DNA from two different sources (the gene from one organism and the plasmid from a bacterium), it is called recombinant DNA.

The full sequence in order is:

1. Cut out the desired gene with a restriction enzyme (leaving sticky ends).
2. Cut open the plasmid with the same restriction enzyme (leaving complementary sticky ends).
3. The matching sticky ends pair up.
4. DNA ligase joins the gene into the plasmid.
5. This forms recombinant DNA, which can be put into a bacterium.

Exam tip. Always name DNA ligase as the joining enzyme (not 'restriction enzyme'). Stating that the product is recombinant DNA earns an extra mark on higher-tariff questions.