Amplifying a gene by PCR

A photocopier for DNA

A gene is a few thousand base pairs. A human cell has about three billion base pairs in a set of chromosomes. To work with one gene, you need many copies of it. The polymerase chain reaction, or PCR, is a machine for that. It copies a chosen region of DNA again and again, in a small tube, in a few hours.

What goes into the tube

• The template: the DNA that holds the wanted gene. It can be very small in amount.
• Two primers. A primer is a short piece of single-stranded DNA, made in the laboratory. Each primer is complementary to a region at one end of the wanted piece, one on each strand.
• The four nucleotides, the building blocks of the new DNA.
• A DNA polymerase, which extends the primers. The enzyme that is used is a thermostable one: it is not destroyed at high temperature. It comes from a bacterium called Thermus aquaticus that lives in hot springs. The enzyme is called Taq polymerase.

The three steps of one cycle

1. Denaturation. The tube is heated to about 94 degrees Celsius. The two strands of the template separate.
2. Annealing. The tube is cooled to about 55 degrees Celsius, and the primers pair with the single strands. The exact temperature depends on the primers.
3. Extension. The tube is warmed to 72 degrees Celsius, the best temperature for Taq polymerase. The enzyme adds nucleotides at the end of each primer, using the strand as the template. Now there are two double-stranded molecules where there was one.

These temperatures are standard values. NCERT describes the three steps but does not give the numbers. A machine called a thermocycler goes through the three temperatures automatically.

The arithmetic of cycles

The new strands themselves become templates in the next cycle. So the number of molecules doubles in each cycle, if every molecule is copied.

• After 1 cycle: 2 molecules.
• After 2 cycles: 4. After 3 cycles: 8.
• After $n$ cycles: $2^n$ molecules.
• After 30 cycles: $2^{30}$, which is a little more than one billion (1 073 741 824). NCERT says that the segment can be amplified to about a billion times.

In a real tube, a cycle copies a little less than all the molecules. If the efficiency of a cycle is $e$, where $e = 1$ means a perfect cycle, the number after $n$ cycles is $N_0 (1 + e)^n$. With an efficiency of 0.9, 30 cycles give $1.9^{30}$, which is about 230 million times the start, not a billion.

A point for A-Level and IB

This part is beyond NCERT and the NTA syllabus, but A-Level and IB ask it. The first products are not yet the exact wanted length. The first molecules with both ends fixed by the two primers appear in the third cycle. Starting with one template, the number of molecules of exactly the wanted length after $n$ cycles is $2^n - 2n$. That is 0 after 2 cycles, 2 after 3, and 8 after 4. After many cycles nearly all the molecules have this length, so the unwanted long ones do not matter.

Why PCR is useful

The amplified gene can be used to ligate into a vector for cloning. PCR is also used in the diagnosis of disease, and in the study of old or very small samples. The next chapter of this book takes up these uses.

You should see that the log of the copies is a straight line against the number of cycles. A small fall in the efficiency makes a big difference after 30 cycles.

Try it in Python

The program counts the molecules in the tube cycle by cycle. It also counts the molecules of exactly the wanted length.

Practise with new numbers

Common mistakes

• Mixing up the three steps. Denaturation separates the strands, annealing pairs the primers, and extension builds the new strands.
• Saying that PCR copies DNA with the ordinary enzyme of a cell. The enzyme must survive about 94 degrees Celsius. It is Taq polymerase.
• Adding instead of multiplying. The number doubles in each cycle, so after 30 cycles it is a billion, not 60.
• Thinking that primers are made by the cell. They are short pieces of DNA that are made in the laboratory.
• Forgetting the two primers. One is needed at each end of the wanted region, one for each strand.

Remember these

• PCR needs a template, two primers, nucleotides and Taq polymerase, from Thermus aquaticus.
• The steps: denaturation (about 94 degrees Celsius), annealing (primers pair, about 55 degrees Celsius) and extension (72 degrees Celsius).
• Ideal copies after $n$ cycles: $2^n$. About one billion after 30 cycles.

Check yourself

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

1. Why is Taq polymerase used in PCR, and where does it come from?

2. What happens in each of the three steps of one cycle?

3. How many molecules are there after 8 cycles, if one template starts and every cycle doubles?

4. About how many cycles give a billion copies from one molecule?

5. Why are two different primers needed?

References

• NCERT (2023). Biology, Textbook for Class XII, chapter 9, Biotechnology: Principles and Processes.
• Mullis K. B. and Faloona F. A. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology 155, 335 to 350.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.2.