DNA Structure and Sequencing

Core Concepts

Nucleotides: the building blocks

Each nucleotide consists of a phosphate group (which gives DNA its negative charge), a five-carbon sugar (deoxyribose; the carbons are numbered 1′–5′, and "deoxy" means it lacks an –OH at the 2′ carbon), and a nitrogenous base attached to the 1′ carbon. Nucleotides join into a strand by phosphodiester bonds between the 5′ phosphate of one nucleotide and the 3′ –OH of the next. The chain therefore has a 5′ end (free phosphate) and a 3′ end (free hydroxyl). This directionality is essential — polymerases build DNA only in the 5′ → 3′ direction, a rule that drives the entire replication story.

Purines and pyrimidines

The four bases come in two shapes. Purines (adenine, guanine) have two fused rings; pyrimidines (thymine, cytosine) have one. In the double helix a purine always pairs with a pyrimidine, which keeps the two sugar-phosphate backbones a constant distance apart. Pairing is specific and held by hydrogen bonds: A pairs with T (two hydrogen bonds) and G pairs with C (three). Because G–C pairs have one more hydrogen bond, G–C-rich DNA is slightly harder to separate — a fact exploited in PCR and melting-temperature calculations.

The double helix

Watson and Crick's 1953 model (built on Franklin's X-ray data and Chargaff's rules) described DNA as two antiparallel strands — one running 5′ → 3′, the other 3′ → 5′ — wound around a common axis. The sugar-phosphate backbones form the outside; the paired bases stack inside like the rungs of a twisted ladder. Commonly taught dimensions: the helix is about 2 nm in diameter, bases are stacked about 0.34 nm apart, and there are about 10 base pairs per turn. The strands are complementary — the sequence of one determines the sequence of the other — which is the basis of copying and hybridization.

DNA versus RNA

RNA differs from DNA in three ways: its sugar is ribose (with an –OH at the 2′ carbon), it uses uracil (U) in place of thymine (U pairs with A), and it is usually single-stranded (though it folds into complex shapes like tRNA cloverleaves and ribosomes). RNA's extra hydroxyl makes it less stable than DNA — a trade-off that suits its many short-lived roles.

Packaging the genome

A human cell's DNA is roughly 2 meters long if fully extended (a commonly taught figure) yet fits into a nucleus about 6 µm across — a compression problem solved by wrapping DNA around histone proteins to form nucleosomes, which coil into chromatin and condense into chromosomes during division. The human genome contains about 3 billion base pairs per haploid set, with roughly 20,000–25,000 protein-coding genes (commonly taught estimates; the exact count is still being refined). Additional twisting (supercoiling) further compacts DNA and is managed by enzymes called topoisomerases.

Sequencing DNA

Sanger (chain-termination) sequencing reads a DNA molecule by copying it in the presence of dideoxynucleotides (ddNTPs). A ddNTP can be added to a growing strand but, lacking the 3′ –OH, cannot be extended — it terminates the chain. With fluorescently labeled ddNTPs, each base produces a chain of a characteristic length, and separating the fragments by size while reading the colors reveals the sequence. Next-generation sequencing (NGS) modernized the idea with massively parallel sequencing-by-synthesis: millions of short fragments are anchored to a surface and read base-by-base as fluorescent signals, generating billions of reads per run that computers assemble into genomes. The same technology powers clinical panels, tumor profiling, and metagenomics.

Common Confusions