10 min readPathway sheetOften a 3-mark answer on the three steps of PCR; 1-mark MCQ on Taq polymerase or the number of copies; also a NEET favourite

Amplifying a gene: PCR

The polymerase chain reaction copies a gene in vitro using two primers and a heat-stable Taq polymerase. Each cycle of denaturation, annealing and extension doubles the DNA.

PCRPolymerase chain reaction: making many copies of a chosen DNA segment in vitro (outside a living cell)

PrimersSmall, chemically synthesised oligonucleotides complementary to regions of the DNA at each end of the target

Taq polymeraseThermostable DNA polymerase from the bacterium Thermus aquaticus; stays active at high temperature

CopiesFrom one DNA, copies after n cycles = 2ⁿ; about 30 cycles give about a billion copies

What a PCR needs

PCR makes many copies of a gene of interest in a tube. It needs the template DNA and two primers. Primers are short single-stranded DNA pieces, made in the lab, that match the two ends of the target region. It also needs the four deoxyribonucleotides (building blocks of DNA) and a thermostable DNA polymerase. A machine changes the temperature in cycles. The polymerase extends the primers using the nucleotides and the template.

One cycle of PCR

Your textbook names the three steps; the temperatures given here are typical laboratory values.

1
Denaturation: heat to a high temperature (about 94°C). The double-stranded DNA separates into two single strands.
High temperature breaks the hydrogen bonds between bases
2
Primer annealing: cool (to about 50 to 60°C). Each primer binds to its complementary sequence on one single strand.
3
Extension: at about 72°C, the polymerase adds nucleotides to each primer, copying the template strand.
Taq polymerase
4★
End of the cycle: two double-stranded copies from one. Repeat the cycle many times.

★ key step

Why Taq polymerase

Every cycle begins with a high-temperature step. An ordinary DNA polymerase would be destroyed at that temperature. Taq polymerase, from Thermus aquaticus, a bacterium of hot springs, stays active during denaturation. So the cycles can be repeated again and again. The amplified gene can then be ligated into a vector for cloning.

How many copies?

Problem

A PCR starts with one double-stranded DNA molecule. How many copies are there after (a) 5 cycles (b) 30 cycles?

Working

Each cycle doubles the number of DNA molecules, so copies = 2ⁿ after n cycles.
(a) 2⁵ = 2 × 2 × 2 × 2 × 2 = 32.
(b) 2³⁰ = 2¹⁰ × 2¹⁰ × 2¹⁰ = 1024 × 1024 × 1024.
1024 × 1024 = 1,048,576; × 1024 = 1,073,741,824, which is about 1.07 × 10⁹.

Answer

(a) 32 copies; (b) about 1.07 × 10⁹ copies, roughly a billion, as your textbook states.

The PCR cycle

back to Double-stranded DNA

How to draw the steps of PCR

Draw a short double-stranded DNA at the top and bracket the region to be amplified.
Below it, draw the two strands pulled apart: label 1. Denaturation at high temperature.
Draw short arrows (primers) bound at the 3′ end of each separated strand: label 2. Annealing of primers.
Draw new strands growing from each primer as dashed lines: label 3. Extension: Taq polymerase, nucleotides.
Draw two double-stranded copies at the bottom and write repeated about 30 times: about 1 billion copies.

In real life

PCR made the RT-PCR test for COVID-19 familiar to every Indian family. The same method detects HIV, the tuberculosis bacterium and gene mutations long before symptoms appear.

Exam angle

3 marks: “Describe the three steps of a PCR cycle. Why is Taq polymerase used?” Denaturation: high temperature separates the strands. Annealing: primers bind to complementary ends. Extension: polymerase adds nucleotides to the primers. Taq polymerase from Thermus aquaticus is thermostable and survives the denaturation temperature, so cycles can repeat.

2 marks: “What are primers? Why are two primers needed in PCR?” Small, chemically synthesised oligonucleotides complementary to regions of the DNA. Each strand needs its own primer, as the polymerase can only extend a primer bound to the template.

1 marks: “How many DNA copies form from one molecule after 10 PCR cycles?” 2¹⁰ = 1024 copies.

Remember it

PCR goes D-A-E: Denature (hot), Anneal (cool), Extend (warm), then do it again.