Molecular Diagnostic Techniques

Study Notes

Polymerase Chain Reaction (PCR):
- Useful for detecting a wide range of pathogens, adaptable through various modifications.
- Suitable for diverse sample types.
- Highly sensitive and specific compared to antibody/antigen detection methods.
- Disadvantages: Contamination can lead to false positives; it's more expensive than serological methods; it requires quality control.

FISH: Identifies specific DNA sequences/genes on chromosomes.
- A process used for detection of specific DNA sequences or regions on chromosomes.
- Cells are treated with mild detergent and alkali to prepare them for the procedure.
- Fluorescent probes are labelled and target known sequences during the procedure.
- Specificity in FISH is achieved through probes designed for targeted chromosomes or chromosomal sections.
FISH procedure:
- Cells are prepared in a metaphase stage.
- Fixed on a slide.
- Treated with mild detergent and alkali.
- Fluorescent probes are added and amplified using PCR.
- Hybridized probes are viewed under a microscope for the detection of chromosomal regions.
Probe application: Allows for the use of different coloured fluorescent dyes to distinguish between various DNA sequences on a single chromosome if multiple sequences are being targeted.
Post hybridization process: Samples are washed to eliminate unbound probes and are viewed under fluorescent microscopes for gene identification.
- Amplification, deletion, or duplication of genes can be easily identified using this procedure.

Advantages:
- Detects nucleic acids and antibodies.
- High sensitivity.
- Simultaneous detection of multiple pathogens.
Disadvantages:
- Expensive for routine use.
- Typically requires amplification of nucleic acids prior to analysis.

Sequencing:
- Next-generation sequencing (NGS) is a method that is crucial in genomic profiling of cancer and is used in tailoring treatment plans based on the unique genetic composition of each patient's tumour.
- Biopsies, genetic tests, and biomarker tests are part of this type of analysis.

Description: Laboratory procedures that use tissue, blood, or other body fluids to identify certain genes, proteins, or other molecules related to diseases like cancer—as possible indicators.
Examples: HER2 in breast cancer or EGFR in lung cancer.

Importance: These tests aid in assessing treatment effectiveness.
- Decreasing or normal tumour marker levels suggest treatment efficacy, while increasing levels may indicate treatment resistance or cancer recurrence.
- Chemotherapy frequently causes a temporary increase in tumour marker levels due to the rapid cancer cell death and release of these markers.
Limitations:
- Not sufficient for screening or diagnosing cancer on their own.
- Need to integrate other tests, such as a thorough medical history, physical examination, and other lab tests, for more accurate diagnosis and treatment decisions.
- Some tumour markers may be elevated in individuals without cancer, and various cancer types may share the elevated expression of certain markers, which could lead to misdiagnosis.
- Tumour marker levels may rise only after cancer worsens. This makes it challenging to detect cancer early or determine if cancer has returned after treatment