Molecular Diagnosis

• Early diagnosis and understanding of a disease's pathophysiology are essential for effective medical treatment.
• Normally, a pathogen is only suspected after symptoms appear, by which time the pathogen concentration in the body is already dangerously high.

Core Principle: PCR facilitates early detection by amplifying the nucleic acids (DNA/RNA) of pathogens. It is highly sensitive and can detect extremely low amounts of DNA or low concentrations of bacteria and viruses.

Key Medical Applications:

• Routinely used to detect HIV in suspected patients.
• Used to detect specific gene mutations in suspected cancer patients.
• Utilised in the diagnosis of numerous other genetic disorders.

The Probe: A single-stranded DNA or RNA molecule is tagged with a radioactive marker.

• Important Marker: 32P is commonly used and is incorporated into the phosphodiester bond of the probe DNA.

Hybridisation: This radioactive probe is allowed to hybridise (bind) to its complementary DNA sequence within a clone of cells.

Detection: The results are visualised on a photographic film using a technique called autoradiography.

Identifying Mutations: A cell containing a mutated gene will not be observed on the photographic film. This is because the radioactive probe lacks complementarity with the mutated gene sequence and fails to bind to it.

• Conventional serum and urine analyses are ineffective for early disease detection, making modern molecular methods essential for effective medical treatment.
• Advanced techniques like Polymerase Chain Reaction (PCR), ELISA, and recombinant DNA technology allow for the diagnosis of diseases even before visible symptoms appear.
• PCR facilitates early detection by amplifying the nucleic acids of pathogens, enabling the identification of extremely low concentrations of viruses like HIV.
• PCR is additionally utilised as a highly effective tool to detect specific gene mutations in suspected cancer patients and to diagnose various genetic disorders.
• During autoradiography, a radioactive probe hybridises to complementary DNA; mutations are identified because the probe fails to bind to the mutated sequence, leaving it unobserved on the photographic film.