Laboratory Tools: PCR and Gel Electrophoresis

The polymerase chain reaction (PCR) is a laboratory method that copies a chosen section of DNA millions of times by repeated cycles of heating and cooling, and gel electrophoresis is a method that separates DNA fragments by size using an electric field. In brief: in PCR, each cycle has three steps: denaturation (about 95 °C, strands separate), annealing (about 50 to 65 °C, primers bind) and extension (about 72 °C, a heat-stable DNA polymerase builds new strands). The number of copies doubles with each cycle, so n cycles give about 2ⁿ copies per starting molecule. In electrophoresis, DNA is negatively charged and moves toward the positive electrode through a gel, with the smallest fragments moving fastest and furthest.

PCR and electrophoresis are used in medicine, forensics, research and agriculture. This guide explains the components and steps of PCR, how electrophoresis works and is interpreted, compares the methods, works through a PCR amplification calculation and a gel-reading example, and corrects common mistakes.

What is PCR?

PCR amplifies a specific DNA sequence from a tiny sample, even a single molecule, into amounts large enough to study. It needs these ingredients:

• Template DNA containing the target sequence.
• Two primers: short single-stranded DNA pieces (about 18 to 25 nucleotides) that are complementary to the sequences at each end of the target on opposite strands. They define the region that is copied.
• A heat-stable DNA polymerase: Taq polymerase, from the bacterium Thermus aquaticus from hot springs, which is not destroyed at 95 °C.
• Free nucleotides (dNTPs) as building blocks.
• Buffer and magnesium ions, which provide the right conditions.

The reaction is carried out in a thermal cycler, a machine that changes the temperature precisely and rapidly.

What happens in a PCR cycle?

1. Denaturation (about 94 to 98 °C, 20 to 30 seconds): heat breaks the hydrogen bonds, so the double-stranded DNA separates into two single strands.
2. Annealing (about 50 to 65 °C, 20 to 40 seconds): the temperature is lowered, and the primers bind to their complementary sequences on the template strands. The temperature is chosen to make the binding specific.
3. Extension (about 72 °C, about 1 minute per 1 000 bases): Taq polymerase adds nucleotides to the 3′ end of each primer and makes a new complementary strand.

The cycle is repeated typically 25 to 35 times. After each cycle, the number of copies of the target doubles. In the first cycles, the new strands are of different lengths, but from the third cycle onward the fragments that are of exactly the length between the two primers appear and increase exponentially. In real-time (quantitative) PCR, fluorescent dyes allow the amount of product to be followed as the reaction proceeds. RT-PCR starts with RNA, which is copied into DNA by reverse transcriptase. It is the method used to detect RNA viruses such as SARS-CoV-2.

How does gel electrophoresis work?

1. Prepare the gel: agarose (from seaweed) is dissolved in a buffer, heated, and poured into a mold with a comb to form small wells. It sets into a gel with a mesh of pores. A higher agarose percentage (such as 2 percent) separates small fragments, and a lower percentage (0.8 percent) separates large fragments. Polyacrylamide gels are used for very small fragments, such as in protein separation and DNA sequencing.
2. Load the samples: DNA samples mixed with a loading dye (which makes them dense and visible) are placed into the wells. One well contains a DNA ladder, a mixture of fragments of known sizes.
3. Run the gel: the gel is immersed in buffer, and an electric field (e.g. 100 volts for 30 to 60 minutes) is applied. DNA has a negative charge on its phosphate groups, so the fragments move toward the positive electrode (anode). Short fragments pass through the pores more easily than long ones and move further, which separates DNA by size.
4. Visualize: the DNA is stained with a dye that fluoresces under UV or blue light (such as ethidium bromide, which is a hazardous chemical, or safer alternatives), and bands are seen. Each band is a group of fragments of the same size.
5. Interpret: compare the positions of the bands with the ladder to estimate sizes.

The distance moved is approximately proportional to the logarithm of the size of the fragment, so a graph of log(size) against distance for the ladder is a straight line that can be used to read off the size of unknown fragments.

PCR and electrophoresis compared

Worked example: amplification and reading a gel

Amplification

A PCR starts with 10 copies of the target DNA. Assume 100 percent efficiency.

1. After 10 cycles: 10 × 2¹⁰ = 10 × 1 024 = 10 240 copies.
2. After 20 cycles: 10 × 2²⁰ = 10 × 1 048 576 = 10 485 760, about 10⁷ copies.
3. After 30 cycles: 10 × 2³⁰ = 10 × 1 073 741 824 ≈ 1.07 × 10¹⁰ copies.

If the cycle takes 2 minutes, 30 cycles take 60 minutes, and a ten-billion-fold amplification results. In practice, the efficiency is less than 100 percent (say 90 percent, so that the factor per cycle is 1.9, not 2). Then after 30 cycles: 10 × 1.9³⁰ = 10 × 2.3 × 10⁸ = 2.3 × 10⁹. The reaction also levels off in later cycles because reagents are used up.

Reading a gel

A DNA ladder has fragments of 3 000, 2 000, 1 500, 1 000, 500 and 250 bp. A sample from a PCR run shows two bands, one at the level of the 1 000 bp ladder band and one at the level of the 500 bp band, and another sample shows a single band at the 1 000 bp position. A third sample shows no bands.

1. Sample 1: two fragments of about 1 000 bp and 500 bp. This might be a heterozygous individual with two alleles of different lengths, or the product of a digest with a restriction enzyme.
2. Sample 2: one fragment of 1 000 bp. This might be a homozygote for the longer allele.
3. Sample 3: no band. Possible reasons are that there was no template DNA, that the PCR failed, that the target was absent or that DNA was not loaded. A control sample with the known target should be used to tell the difference.

This kind of analysis is the basis of paternity tests, diagnosis of genetic disease (for instance, finding the deletion in the CFTR gene) and detection of a pathogen. Samples must be compared with controls to avoid false results, especially because PCR is extremely sensitive and can amplify contaminating DNA.

What are the uses?

• Medicine: diagnosing infections (HIV, tuberculosis, COVID-19) and genetic diseases, and testing for mutations in tumors.
• Forensics: comparing DNA from a crime scene with suspects, which is described in the article on DNA profiling.
• Research: cloning genes, preparing DNA for sequencing, studying gene expression.
• Agriculture and food: detecting GMOs, pathogens and food fraud.
• Ancient DNA and ecology: amplifying DNA from fossils, and detecting species from traces of DNA in water (environmental DNA).

What are the limitations and precautions?

PCR needs knowledge of the sequences at the ends of the target to design primers. Its high sensitivity makes contamination a major problem, so work is done with clean technique, separate work areas and negative controls. Taq polymerase lacks proofreading, so errors occur at a low rate (about 1 per 10 000 to 100 000 bases), and high-fidelity polymerases are used when accuracy matters. Electrophoresis gives size information, not sequence. Safety measures include care with electrical equipment, UV light and DNA stains.

How were the methods developed?

Kary Mullis conceived PCR in 1983 while working at Cetus Corporation in California, and the method was made practical by the use of Taq polymerase in 1988, replacing a polymerase that was destroyed by heat in every cycle. Mullis received the Nobel Prize in Chemistry in 1993. Gel electrophoresis for DNA was developed in the 1960s and 1970s, adapting methods that had been used for proteins. Arne Tiselius received the 1948 Nobel Prize in Chemistry for the development of electrophoresis.

Which mistakes are common?

Mistake 1: Thinking PCR needs living cells

PCR is a test-tube reaction with purified ingredients. No cells are involved.

Mistake 2: Mixing up the order of steps

The cycle is denaturation, annealing and extension.

Mistake 3: Saying that DNA moves toward the negative electrode

DNA is negatively charged, so it moves toward the positive electrode.

Mistake 4: Believing that larger fragments run further

Smaller fragments run further, because they move through the gel pores more easily.