14.2 DNA Structure and Sequencing

DNA Structure

The Double Helix: Nucleotide Components and Base Pairing

DNA (deoxyribonucleic acid) is a double-stranded helical molecule. Two polynucleotide chains coil around each other to form a right-handed double helix, and the two strands run in opposite directions. This antiparallel arrangement means one strand runs 5' to 3' while the other runs 3' to 5'.

Each polynucleotide chain is built from repeating units called nucleotides. A single nucleotide has three components:

• A nitrogenous base: adenine (A), thymine (T), guanine (G), or cytosine (C)
• A deoxyribose sugar (a five-carbon sugar missing one oxygen compared to ribose)
• A phosphate group

Nucleotides are linked together by phosphodiester bonds that form between the 5' phosphate group of one nucleotide and the 3' hydroxyl group of the next. This creates the sugar-phosphate backbone that runs along the outside of the helix.

The two strands are held together by complementary base pairing between the nitrogenous bases on opposite strands:

• Adenine (A) pairs with Thymine (T) through two hydrogen bonds
• Guanine (G) pairs with Cytosine (C) through three hydrogen bonds

Because G-C pairs have three hydrogen bonds instead of two, regions of DNA rich in G-C pairs are more stable and harder to separate than A-T rich regions. Beyond hydrogen bonding, hydrophobic interactions between the stacked bases also help stabilize the helix.

This complementary base pairing is what makes accurate DNA replication and transcription possible. If you know the sequence of one strand, you automatically know the sequence of the other.

DNA Sequencing

Steps in Sanger Sequencing

Sanger sequencing (also called chain-termination sequencing) reads the nucleotide sequence of a DNA fragment. The method relies on modified nucleotides that stop DNA synthesis at specific bases, generating fragments of every possible length.

1. DNA denaturation: The double-stranded DNA template is heated to separate it into single strands.

2. Primer annealing: A short oligonucleotide primer, complementary to a specific region of the template, is added. It binds to the single-stranded DNA and gives DNA polymerase a starting point.

3. Extension and termination:

   • DNA polymerase extends the primer, building a new complementary strand.
   • The reaction mixture contains all four normal deoxynucleotide triphosphates (dNTPs) plus a small amount of dideoxynucleotide triphosphates (ddNTPs). ddNTPs lack the 3' hydroxyl group needed to form the next phosphodiester bond.
   • Whenever a ddNTP is randomly incorporated instead of a normal dNTP, chain elongation stops. This produces DNA fragments of varying lengths, each ending at a position where that particular base occurs.

4. Gel electrophoresis: The terminated fragments are separated by size using polyacrylamide gel electrophoresis. Shorter fragments migrate faster through the gel.

5. Visualization and sequence determination: Fragments are detected using fluorescence (each ddNTP is labeled with a different fluorescent dye) or autoradiography. Reading the fragments from shortest to longest reveals the DNA sequence.

Polymerase chain reaction (PCR) is often used before sequencing to amplify small DNA samples so there's enough material to work with.

Sanger sequencing was the workhorse behind the Human Genome Project and remains widely used for targeted sequencing of specific genes and for validating results from newer technologies.

Advanced DNA Sequencing Techniques

Next-generation sequencing (NGS) technologies can sequence millions of DNA fragments simultaneously, making whole-genome sequencing far faster and cheaper than Sanger methods. While Sanger sequencing reads one fragment at a time, NGS platforms process massive numbers of reads in parallel.

These high-throughput approaches build on the same principle that DNA replication is semiconservative: each new DNA molecule contains one original strand and one newly synthesized strand. Understanding this replication mechanism is what made both classical and modern sequencing techniques possible.

DNA Packaging

DNA Packaging in Eukaryotes vs. Prokaryotes

Cells face a real space problem: even a single human cell contains about 2 meters of DNA that must fit inside a nucleus roughly 6 micrometers across. Prokaryotes and eukaryotes solve this packaging challenge in different ways.

Prokaryotic DNA organization:

• Prokaryotic cells (bacteria and archaea) typically have a single, circular chromosome located in a region called the nucleoid. This region is not enclosed by a membrane.
• Proteins called nucleoid-associated proteins (NAPs) help compact and organize the DNA, but the packaging is relatively simple because prokaryotic genomes are much smaller (the E. coli genome is about 4.6 million base pairs).

Eukaryotic DNA organization:

• Eukaryotic cells contain multiple linear chromosomes housed within a membrane-bound nucleus.
• Eukaryotic DNA is packaged through several levels of increasing compaction:
  • Nucleosomes: DNA wraps about 1.65 times around a core of eight histone proteins (a histone octamer). The nucleosome is the basic unit of chromatin.
  • Chromatin fibers: Nucleosomes coil and stack together, aided by linker histones, to form a more compact fiber.
  • Higher-order structures: Chromatin fibers fold into loops and domains, organizing genes into functional regions.
  • Chromosomes: During cell division, chromatin condenses dramatically into the compact, visible chromosomes you see in micrographs of mitosis and meiosis.

This multi-level packaging does more than just save space. It also plays a direct role in regulating gene expression: tightly packed chromatin (heterochromatin) tends to silence genes, while loosely packed chromatin (euchromatin) is accessible for transcription. Proper packaging also ensures chromosomes segregate correctly during cell division.