Polymerase Chain Reaction (PCR), gel electrophoresis and their applications

Polymerase Chain Reaction (PCR) and gel electrophoresis are powerful tools used by genetic engineers in DNA analysis.

PCR is a technique that makes millions of copies of a specific DNA segment, helping scientists study even tiny amounts of DNA.

Gel electrophoresis is a method used to visualise the DNA fragments by separating them based on their size and charge by running them through an agarose gel using electricity.

These techniques are widely used in areas like medicine, forensics, and research. Together, they allow scientists to better understand and work with DNA.

Use this page to revise the following concepts:

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify a specific segment of DNA in a sample by generating millions or billions of copies from a small starting amount. PCR is widely used in research, medicine, forensics, and biotechnology due to its speed, accuracy, and ability to work with tiny amounts of DNA. PCR is a method of artificial DNA replication.

What is required for PCR?

  1. DNA sample - contains the segment to be amplified.
  2. Primers - Short pieces of single-stranded DNA that are complementary to the target DNA sequence.
  3. Nucleotides - building blocks (A, T, G, C) used to create new DNA strands.
  4. Taq DNA polymerase - enzyme that synthesizes new DNA strands.
  5. Buffer solution - maintains the optimal conditions for the reaction.
  6. Microtest tube - where all of the above are combined, and the reaction occurs.

The PCR process involves three main steps, which are repeated in cycles.

  1. Denaturation (94-98oC):
    The double-stranded DNA is heated to a high temperature to break the hydrogen bonds between the complementary bases, causing the DNA to separate into two single strands. This step prepares the DNA for binding to the primers.
  2. Annealing (50–65°C):
    The reaction mixture is cooled to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA template.
  3. Extension (72°C):
    The temperature is raised to the optimal working temperature of Taq DNA polymerase. Starting from the primers, the polymerase synthesises new DNA strands by adding nucleotides complementary to the template strand.

This process doubles the amount of targeted DNA in the reaction, so that at the end of one cycle there are two identical DNA molecules.

The diagram below shows one cycle of PCR.

The three step above process is repeated 25 - 30 times, resulting in millions of copies of the DNA, as each cycle doubles the amount of DNA.

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate and analyse DNA, RNA, or protein fragments based on their size and charge. It works by applying an electric field to an agarose gel, which causes charged molecules to move through the gel. Smaller molecules will move more quickly through the gel than larger molecules. The separation of molecules helps scientists identify specific sequences or determine the size of fragments.

How gel electrophoresis works: 

  1. An agarose gel is prepared. The gel is typically prepared in a gel tray and submerged in a buffer solution that maintains the pH and ensures electrical conductivity. A comb is put in the unset gel to create wells, where the DNA will be loaded.
  2. DNA samples together with a loading dye (which helps track the progress of the DNA as it moves), are loaded into the wells of the gel.
    A DNA ladder (a set of DNA fragments of known sizes) is typically loaded into one well to help estimate the sizes of the DNA fragments in the samples.
  3. The gel is placed in an electrophoresis chamber, and an electric current is applied. DNA molecules are negatively charged due to their sugar - phosphate backbone, so they will move toward the positive electrode (the anode).
  4. The rate at which the DNA moves through the gel depends on its size —smaller DNA fragments move faster while larger fragments move more slowly through the pores in the gel.
  5. After the electrophoresis run is complete, the gel is stained with a dye that binds to DNA and fluoresces under ultraviolet (UV) light.

The bands can be visualized under UV light, allowing scientists to analyse the DNA fragments.

Applications and Uses

Gel electrophoresis and PCR are powerful techniques widely used in molecular biology and biotechnology for analysing and manipulating DNA. They have various applications in fields such as genetics, forensics, medical diagnostics, and research, enabling scientists to identify genetic markers, detect diseases, and study gene sequences with high precision.

Here are examples of when PCR and gel electrophoresis are used:

  1. Forensics: Often, at a crime scene, there is only a very small amount of DNA left on a weapon or a victim. To be able to analyse this DNA, PCR is used to create millions of copies of this DNA, so analysis can be carried out. The DNA can then be compared to known criminals, or the DNA can be stored in a DNA databank.

  2. Mass disasters: Unfortunately, there are natural and human induced disasters where the victims can not be identified using usual visual methods, as their bodies are beyond recognition. Examples include bombings, building collapses, and earthquakes.
    To identify these individuals, samples of DNA are taken from the deceased victims and amplified using PCR to have multiple copies of the DNA. This DNA is compared to DNA of surviving relatives and any artefacts that might have been found around the disaster area that could contain DNA, such as toothbrushes and hairbrushes.
  3. Genetic diseases: PCR and gel electrophoresis can be used to determine which family members carry a specific allele associated with a disease. For example, individuals with certain alleles of the BRCA2 gene (Breast cancer gene 2) have a predisposition to breast cancer and other cancers associated with the BRCA2 gene. If an individual is found to carry the allele, they can choose whether they take preventative actions, such as a mastectomy.
  4. Paternity testing: If a parent of a child is unknown, DNA samples can be taken from suspected parents to establish paternity.