Medical Genetics · MU Plovdiv

6. DNA analysis. PCR. Southern blot. Mutation analysis. DNA sequencing.

Inside: 13 sections · 13 labelled pictures · 1 table · 41 flashcards · 11 min

Topic 6 of 33 in the MU Plovdiv syllabus for Medical Genetics (Medicine, Year 4). The free AceMed note covers it in 12 sections, about 1,593 words (11 min of reading), with 41 flashcards.

In this note

  1. Introduction
  2. Electrophoresis
  3. Polymerase chain reaction (PCR)
  4. Reverse transcription PCR (RT-PCR)
  5. Allele-specific PCR
  6. Quantitative fluorescence PCR (QF-PCR)
  7. Real-time PCR (qPCR)
  8. Blotting techniques
  9. Restriction fragment length polymorphism (RFLP) analysis
  10. Allele-specific oligonucleotides (ASO)
  11. DNA sequencing
  12. Clinical and research applications

Introduction

Molecular genetic methods are laboratory techniques that allow detection, amplification and analysis of specific DNA sequences. This topic covers electrophoresis, PCR and its variants, blotting (Southern blot), mutation analysis (allele-specific PCR, RFLP, ASO) and DNA sequencing (Sanger and next-generation).

Introduction

  • 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.

Electrophoresis

Electrophoresis separates DNA fragments by size by applying an electric field.

  • Principle: charged molecules move in an electric field. DNA is negatively charged (phosphate backbone) and migrates towards the positive electrode (anode).
  • Method: DNA samples are loaded into wells in a gel matrix, often agarose, and current is passed through the gel. Smaller fragments move faster, larger fragments more slowly, so they separate by size.
  • Visualizing: the gel is stained with a fluorescent dye (ethidium bromide) that binds DNA; under UV light the fragments appear as bright bands. Position and intensity are compared with molecular weight markers to assess size and concentration.
  • Use in PCR analysis: the PCR product is run on a gel; if amplification worked, a band appears at the expected size.
  • Mnemonic: "DNA Moves Towards Positive Small Spots": DNA is negatively charged, moves towards the positive electrode, smaller fragments move faster, stained with ethidium bromide.

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