Choosing enzymes from a restriction map

Plan first, cut later

This lesson goes beyond NCERT. It is the kind of planning that A-Level, IB and Olympiad questions test. The idea is simple. The enzymes are chosen on paper, from a map. Then the experiment in the laboratory works the first time.

Reading a restriction map

A restriction map shows the places where enzymes cut a DNA. It is drawn as a line for a straight DNA and as a circle for a plasmid. The distance between two sites, in base pairs, is the size of the fragment between them. For a straight DNA with $k$ sites, there are $k + 1$ fragments. For a circle with $k$ sites, there are $k$ fragments.

Choosing enzymes: three rules

1. Do not cut inside the gene. If an enzyme has a site inside the gene, it cuts the gene in pieces. Choose enzymes whose sites lie outside the part that you want.
2. Cut the vector once or twice, in its cloning region. The sites should be unique in the vector.
3. Make the ends fit. The insert and the vector must have fitting ends. Cut both with the same enzymes, or with enzymes that leave the same overhang.

One enzyme, or two?

With one enzyme, both ends of the insert are the same, and both ends of the opened vector are the same. Two things can go wrong.

• The vector can close again without an insert. The ends fit each other.
• The insert can go in either direction. Two kinds of recombinant form.

With two different enzymes, the ends of the insert are different, and the ends of the vector are different. An EcoRI end fits only an EcoRI end. A BamHI end fits only a BamHI end. So:

• The insert goes in one direction only. This is directional cloning.
• The vector cannot close on itself, because its two ends do not fit.

Compatible ends

Enzymes with different names can leave the same overhang. BamHI (G^GATCC) and BglII (A^GATCT) both leave GATC. SalI and XhoI both leave TCGA. A BamHI end can be ligated to a BglII end. The joint is a hybrid, and neither enzyme cuts it again. This is a useful fact, and also a trap: you cannot cut out the insert again with either enzyme.

Stopping the vector from closing: phosphatase

When only one enzyme is used, workers treat the cut vector with alkaline phosphatase. This enzyme removes the phosphate group at the 5 prime end of the DNA. Ligase needs this phosphate to seal the backbone. A vector without it cannot close on itself. The insert still has its phosphates, so it can join the vector, and the host repairs the small gaps that are left.

Which way did the insert go?

Suppose the insert went in with one enzyme only. We need to know the direction. We cut the recombinant plasmid with a second enzyme that has one site in the insert, off the centre, and one site in the vector. The sizes of the fragments are different for the two directions.

Here is the data of the program below. The vector has 3000 bp, with an EcoRI site at 400 and a HindIII site at 1000. The insert has 1200 bp and an internal HindIII site 200 bp from its left end. The recombinant has 4200 bp. If the insert goes in the first way, the HindIII sites lie at 600 and at 2200, and the fragments are 1600 and 2600 bp. If it goes in reversed, the sites lie at 1400 and at 2200, and the fragments are 800 and 3400 bp. A gel tells the two apart at once.

You should see that the recombinant is longer than the vector by the insert minus the stuffer. Two enzymes close together cost the vector only a few base pairs.

Try it in Python

The program cuts a vector with two enzymes and builds the recombinant.

Practise with new numbers

Common mistakes

• Choosing an enzyme that cuts inside the gene. The gene is cut in pieces.
• Forgetting the stuffer. The piece between the two sites is lost.
• Saying that one enzyme gives directional cloning. It needs two different enzymes.
• Thinking that BamHI can cut out an insert that was joined to a BglII end. The hybrid joint is not a site of either enzyme.
• Using a diagnostic site in the middle of the insert. The two directions then give the same fragments.

Remember these

• Pick enzymes that cut outside the gene, and once or twice in the cloning region of the vector.
• Two different enzymes: the insert goes one way only, and the vector cannot close on itself.
• BamHI and BglII leave GATC. SalI and XhoI leave TCGA. The hybrid joint is not cut again.
• Alkaline phosphatase stops a vector from closing on itself.
• A diagnostic digest with an off-centre site shows the direction.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Why are two different enzymes better than one for cloning?

2. A vector of 3500 bp is cut with two enzymes whose sites are 70 bp apart. An insert of 1000 bp is ligated in. How long is the recombinant?

3. What does alkaline phosphatase do to a vector, and why is it used?

4. Can BamHI cut a joint made between a BamHI end and a BglII end?

5. Why is an off-centre site used to find the direction of an insert?

References

• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.2.
• Brown T. A. (2016). Gene Cloning and DNA Analysis: An Introduction, 7th edition, Wiley, chapter 5 (Introduction of DNA into living cells).