Ligases, polymerases, vectors and hosts

Joining what was cut

After a restriction enzyme has cut two DNA molecules, we have pieces with sticky ends. Suppose we mix pieces cut by the same enzyme. The sticky ends find each other and pair by hydrogen bonds. But these bonds are weak, and the pieces can fall apart again.

DNA ligase makes the joint permanent. It seals the gaps between the sugar and the phosphate of the two pieces. It acts like molecular glue. The new DNA, made by joining pieces from two sources, is the recombinant DNA.

Two pieces can be joined if their ends fit. Sticky ends fit when their overhangs are complementary. Ends made by one enzyme always fit. Ends made by different enzymes can fit too, if the overhangs happen to match.

Polymerases

DNA polymerases build a new strand by adding nucleotides to a primer, using a strand as a template. Cells use them to copy their DNA, and the host copies the cloned gene with its own polymerase. In the laboratory, a polymerase that does not break down at high temperature is used to make many copies of a gene. This is the PCR of the next course. Polymerases are the second member of the tool kit.

Vectors

A gene cannot enter a cell and copy itself on its own. A piece of DNA has to be carried. The carrier is called a vector, which means a vehicle. The vectors used most often are plasmids and bacteriophages. Both can copy themselves inside a bacterium without being under the control of the chromosome of the host, and they make many copies.

A cloning vector needs the following parts.

• Origin of replication (ori). This is a particular sequence where the copying of DNA starts. Any DNA linked to it is copied too. The ori also controls the copy number, which is how many copies of the vector are in one cell. If you want a lot of the cloned DNA, choose a vector whose ori gives a high copy number.
• Selectable marker. This is a gene that helps us find the cells that took up the vector. It often gives resistance to an antibiotic. The genes used most often are ampR (ampicillin), tetR (tetracycline), chlR (chloramphenicol) and kanR (kanamycin). Normal E. coli cells have no resistance to these antibiotics. So only the cells that took up the vector survive when the antibiotic is in the medium.
• Cloning sites. These are places where a restriction enzyme can cut the vector, so that the foreign DNA can be linked. The vector should have very few such sites, and best of all just one for the enzyme we use. With more than one site, the enzyme would cut the vector into several pieces, and that would make the cloning hard.

The plasmid pBR322

NCERT shows the plasmid pBR322 as an example of a cloning vector. The name has a meaning: p stands for plasmid, B and R stand for the two workers who made it, Bolivar and Rodriguez, and 322 is the number of this plasmid in their laboratory.

It has all three parts.

• the ori;
• two antibiotic resistance genes, ampR and tetR, as selectable markers;
• several cloning sites. NCERT shows the sites of HindIII, EcoRI, BamHI, SalI, PvuII, PstI and ClaI. Some of the sites lie inside the genes: PstI in ampR, and BamHI and SalI in tetR.
• a gene called rop, which codes for proteins that take part in the copying of the plasmid.

The picture shows the order of the parts. It is not drawn to scale. The plasmid has 4361 base pairs. Its sites are placed inside the genes for a reason, and you will see the reason in the next course: a foreign gene put into one of those sites switches the gene off.

The host

The host is the cell in which the work is done. The DNA is large and it likes water. It cannot cross the cell wall and the membrane of a bacterium easily. So the host has to be made ready. A cell that has been treated so that it takes up DNA is called competent. The treatment is described in the next course.

For plants and animals, other vectors are used. The Ti plasmid of the bacterium Agrobacterium tumefaciens is changed to carry genes into plant cells. Disarmed viruses, which cannot cause disease, carry genes into animal cells.

You should see that a vector with a higher copy number gives more copies of the gene, in the same culture. For a culture of the same size, 100 copies in each cell give five times as many gene copies as 20 copies.

Try it in Python

The program tells which sticky ends can be joined. It works out the overhang of each enzyme from its site.

Practise with new numbers

Common mistakes

• Saying that ligase cuts DNA. Ligase joins. Restriction enzymes cut.
• Thinking that hydrogen bonds between sticky ends are enough. They hold the pieces for a short time. Ligase makes the bond permanent.
• Saying that a good vector has many cloning sites. It should have very few, best of all one.
• Reading pBR322 as a 322 base pair plasmid. The number is a serial number of the plasmid. It has 4361 base pairs.
• Mixing up the roles. The ori makes copies, the marker finds the cells that took it up, and the cloning site receives the gene.

Remember these

• DNA ligase seals the sugar-phosphate backbone and makes a joint permanent.
• A vector has an origin of replication, a selectable marker and cloning sites. Few cloning sites, best of all one.
• Markers: ampR, tetR, chlR, kanR. Normal E. coli has none of them.
• pBR322: ori, ampR, tetR, rop and cloning sites. Sites in tetR: BamHI, SalI. A site in ampR: PstI.
• A host must be made competent to take up DNA.

Check yourself

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

1. What does DNA ligase do, and why is it needed after the sticky ends have paired?

2. Name the three features of a cloning vector, and give one job of each.

3. Why should a vector have only one site for the enzyme that is used?

4. What does each part of the name pBR322 stand for?

5. Each cell of a culture has 40 copies of a plasmid. How many copies are there in 300 million cells?

References

• NCERT (2023). Biology, Textbook for Class XII, chapter 9, Biotechnology: Principles and Processes.
• Bolivar F. et al. (1977). Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2, 95 to 113.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.2.