Genetics · MU Sofia

18. Molecular genetic methods: DNA/RNA isolation, liquid biopsy, variant nomenclature, PCR-based techniques, qPCR, ddPCR, MLPA, repeat expansion analysis, Southern blot.

Inside: 15 sections · 9 labelled pictures · 2 tables · 14 flashcards · 8 min

Topic 18 of 79 in the MU Sofia syllabus for Genetics (Medicine, Year 3). The free AceMed note covers it in 14 sections, about 952 words (8 min of reading), with 14 flashcards.

In this note

  1. Molecular Genetic Methods
  2. I. Introduction
  3. II. Polymerase Chain Reaction (PCR)
  4. III. Allele-Specific PCR
  5. IV. Quantitative Fluorescence PCR (QF-PCR)
  6. V. Real-Time PCR (qPCR)
  7. VI. Restriction Fragment Length Polymorphism (RFLP) Analysis
  8. VII. Allele-Specific Oligonucleotides (ASO)
  9. VIII. Clinical and Research Applications
  10. Detection of Deletions and Duplications: MLPA & aCGH
  11. II. MLPA (Multiplex Ligation-dependent Probe Amplification)
  12. III. Molecular Karyotyping (aCGH – Array Comparative Genomic Hybridization)
  13. IV. Comparison: MLPA vs aCGH
  14. V. Clinical Relevance

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.

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