Why Early Diagnosis Matters

For a disease to be treated effectively, two things need to happen early: the disease must be diagnosed, and its pathophysiology — how it disturbs the normal working of the body — must be understood. The trouble with conventional methods such as serum and urine analysis is that they cannot detect a disease early. They pick up trouble only once it has already advanced.

This is where molecular tools change the picture. Recombinant DNA technology, the Polymerase Chain Reaction (PCR) and the Enzyme Linked Immuno-Sorbent Assay (ELISA) all serve the purpose of catching a disease before it has had the chance to do serious damage.

Catching a Pathogen with PCR

Normally, a pathogen such as a bacterium or a virus is suspected only after it has produced a disease symptom. By that time its concentration in the body is already very high. Ideally we would want to spot it much earlier, when its numbers are still tiny and no symptom has appeared.

That early detection is exactly what PCR makes possible. Even a very low concentration of a bacterium or virus can be detected by amplifying its nucleic acid — making millions of copies of the tell-tale DNA until there is enough to detect. In routine practice, PCR is used to detect HIV in suspected AIDS patients and to detect mutations in genes in suspected cancer patients. It is also a powerful technique for identifying many other genetic disorders.

Probes and Hybridisation

A second approach uses the pairing rules of nucleic acids directly. A single-stranded DNA or RNA is tagged with a radioactive molecule — this tagged strand is called a probe. The probe is allowed to hybridise to its complementary DNA in a clone of cells, and the result is read using autoradiography, which records the radioactivity on a photographic film.

The elegant part is what happens when a gene is faulty. A clone carrying a mutated gene will not appear on the film, because the probe is no longer perfectly complementary to the altered sequence and therefore cannot bind to it. Absence of a signal, in other words, flags the mutation.

ELISA and Antigen-Antibody Interaction

The third technique, ELISA, works on the principle of antigen-antibody interaction. When a pathogen infects the body, it can be picked up in one of two ways.

One route is to detect the antigens of the pathogen itself — its proteins, glycoproteins and similar molecules. The other route is to detect the antibodies that the patient's own immune system has synthesised against that pathogen. Either signal tells us the infection is present, which is why ELISA is such a workhorse in clinical laboratories.

Quick Recap

  • Effective treatment needs early diagnosis and an understanding of pathophysiology; conventional methods (serum and urine analysis) cannot detect disease early.
  • Recombinant DNA technology, PCR and ELISA enable early diagnosis.
  • A pathogen is usually suspected only after symptoms appear, when its concentration is already high; PCR detects very low concentrations by amplifying nucleic acid.
  • PCR is used to detect HIV in suspected AIDS patients, mutations in suspected cancer patients, and many genetic disorders.
  • A radioactively tagged probe hybridises to complementary DNA and is read by autoradiography; a mutated gene will not appear on the film because the probe cannot pair with it.
  • ELISA is based on antigen-antibody interaction — detecting either the pathogen's antigens or the antibodies made against it.

Solved Examples — Section 9

Q1. Why are conventional methods of diagnosis inadequate for early detection?

Answer: Methods such as serum and urine analysis cannot detect a disease early; they reveal it only after it has advanced.

Q2. Name three techniques used for early molecular diagnosis.

Answer: Recombinant DNA technology, PCR (Polymerase Chain Reaction) and ELISA (Enzyme Linked Immuno-Sorbent Assay).

Q3. How can PCR detect a pathogen present in very low concentration?

Answer: By amplifying the pathogen's nucleic acid — making many copies of it — so that even very small amounts can be detected before symptoms appear.

Q4. Give two routine clinical uses of PCR in diagnosis.

Answer: Detecting HIV in suspected AIDS patients and detecting mutations in genes in suspected cancer patients (also identifying genetic disorders).

Q5. Why will a clone with a mutated gene not appear on the autoradiography film?

Answer: Because the radioactive probe is not complementary to the mutated gene, so it cannot hybridise with it and no signal is recorded.

Q6. On what principle does ELISA work, and what can it detect?

Answer: It works on antigen-antibody interaction; it detects either the pathogen's antigens (proteins, glycoproteins) or the antibodies synthesised against the pathogen.