Molecular Biology and DNA Sequencing

Introduction to DNA Sequencing

• Significant progress in molecular biology due to DNA sequencing ability.
• Human Genome Project relied on advanced technology derived from basic DNA sequencing methods.
• Dideoxy Sequencing Method: Foundational for DNA sequencing.
• Importance of understanding basic DNA structure.

Basic DNA Structure

• DNA: Deoxyribonucleic Acid.
• Composed of nucleotides:
  • Phosphate group
  • Sugar (Deoxyribose)
  • Nitrogenous base (A, G, C, T)
• Bonding mechanism:
  • Phosphate of one nucleotide bonds to the sugar of another via dehydration synthesis (produces water).
  • Deoxyribose lacks an oxygen in one hydroxyl group.
  • Dideoxyribose lacks oxygen in both hydroxyl groups, preventing bonding.

Dideoxy Sequencing Method

• Uses dideoxynucleotides to stop DNA sequence assembly.
• Four nitrogenous bases: Thymine (T), Cytosine (C), Adenine (A), Guanine (G).
• Experimentation with dideoxynucleotides:
  • Experiment T: Regular A, G, C; dideoxy-T used to stop DNA sequence.
  • Experiment C: Regular A, G, T; dideoxy-C used.
  • Experiment G: Regular A, C, T; dideoxy-G used.
  • Experiment A: Regular G, C, T; dideoxy-A used.

Gel Electrophoresis

• Analyzing DNA sequence length by separating DNA fragments in thick gel.
• Electrical current used to pull DNA through the gel.
• Smaller DNA molecules travel further.
• Lanes labeled according to dideoxynucleotide used.

Reconstructing DNA Sequence

• Analyze sequence based on distance traveled in gel.
• Example sequence determination: TGATT.
• Final position remains ambiguous unless longer segment examined.

Conclusion

• Understanding dideoxy sequencing is crucial for genetics, molecular biology, and genome studies.