I. Introduction
Molecular genetic methods are laboratory techniques that allow detection, amplification, and analysis of specific DNA sequences.
- Applications: diagnosing genetic diseases, identifying carriers, prenatal testing, forensic identification, cancer genetics, and research.
- Core principle: DNA can be amplified, cut, hybridized, and quantified using specific enzymes, primers, and probes.
II. Polymerase Chain Reaction (PCR)
- Definition: Technique to exponentially amplify a specific DNA fragment in vitro.
- Principle: Uses DNA polymerase to replicate a DNA target between two primers through repeated cycles of heating and cooling.
- Steps:
- 1. Denaturation (95°C): Double-stranded DNA unwinds → single-stranded.
- 2. Annealing (50–65°C): Primers bind to complementary target sequences.
- 3. Extension (72°C): Taq DNA polymerase extends primers → new DNA strands.
- Repeated ~25–40 cycles → billions of copies of target DNA.
- Applications:
- Detection of infectious agents (HIV, TB, SARS-CoV-2).
- Genetic testing (cystic fibrosis, BRCA mutations).
- Forensic DNA profiling.
III. Allele-Specific PCR
- Definition: Modified PCR method designed to distinguish between wild-type and mutant alleles differing by only a single nucleotide.
- Principle:
- Primer is designed to end exactly at the mutation site.
- PCR only occurs if primer perfectly matches the DNA sequence.
- Applications:
- Detecting point mutations (e.g., sickle cell anemia, KRAS mutations in cancer).
- Carrier detection in families.
- Clinical example: Differentiating normal β-globin gene vs sickle cell allele.