Overview
This lecture covers the molecular structure of DNA, methods for sequencing DNA—especially the Sanger method—and compares DNA organization in prokaryotes and eukaryotes.
DNA Structure
- DNA is made of nucleotides, each with a nitrogenous base, a 5-carbon sugar, and a phosphate group.
- Nitrogenous bases are purines (adenine, guanine) or pyrimidines (cytosine, thymine).
- In DNA, sugar is deoxyribose; in RNA, sugar is ribose.
- Nucleotides are joined by 5'-3' phosphodiester bonds forming a sugar-phosphate backbone.
- DNA strands are antiparallel and form a right-handed double helix.
- Base pairing: adenine pairs with thymine (2 hydrogen bonds), guanine with cytosine (3 hydrogen bonds).
- DNA double helix has major and minor grooves for protein binding.
- A DNA helix turn is 3.4 nm, includes 10 base pairs, and DNA diameter is 2 nm.
DNA Sequencing Techniques
- Sanger’s dideoxy chain termination method uses dye-labeled ddNTPs to terminate DNA synthesis at specific bases.
- Four reactions (A, T, G, C) generate fragments ending at each base, separated by capillary electrophoresis.
- Each fragment’s end is detected by a fluorescent label, allowing the sequence to be read.
- Gel electrophoresis separates DNA fragments by size; smaller fragments move farther in the gel.
DNA Packaging and Comparison: Prokaryotes vs. Eukaryotes
- Prokaryotes have a single circular chromosome in the cytoplasmic nucleoid region.
- Eukaryotes have linear chromosomes in a membrane-bound nucleus and use histones to pack DNA into nucleosomes.
- DNA in prokaryotes is supercoiled for compaction; in eukaryotes, nucleosomes form higher-order structures (chromatin fibers).
- Eukaryotic chromatin has heterochromatin (densely packed, inactive) and euchromatin (loosely packed, active).
Key Terms & Definitions
- Nucleotide — building block of DNA/RNA with base, sugar, and phosphate group.
- Purine — double-ring nitrogenous base (adenine, guanine).
- Pyrimidine — single-ring nitrogenous base (cytosine, thymine).
- Phosphodiester bond — link between nucleotides’ sugar-phosphate backbone.
- Antiparallel — opposing orientation of DNA strands.
- Dideoxynucleotide (ddNTP) — DNA building block lacking 3' OH, terminates DNA synthesis in sequencing.
- Histone — protein that DNA wraps around in eukaryotes to form nucleosomes.
- Nucleoid — region in prokaryotes where the chromosome is located.
Action Items / Next Steps
- Review DNA structure diagrams and base pairing rules.
- Watch linked videos on Sanger sequencing and genome sequencing.
- Study the differences in DNA packaging between prokaryotic and eukaryotic cells.