Every cell in your body holds about two metres of DNA folded into a nucleus a few micrometres wide, and that DNA carries the instructions to build you. Yet as late as the 1940s most biologists thought genes were made of protein. Proteins seemed the obvious choice: they are built from 20 different amino acids and come in endless shapes, while DNA is built from only four kinds of nucleotide and looked too simple and repetitive to carry information. Overturning that belief took a chain of clever experiments over about 25 years, each one ruling out an alternative. How the evidence was gathered matters as much as the answer, because the same logic (change one thing, label one molecule, see what gets passed on) still drives molecular biology. Once DNA was confirmed as the genetic material, the race was on to find its structure, and the structure immediately suggested how DNA could be copied.
The search for the hereditary material
| Year | Scientist(s) | What they did | What it showed |
|---|---|---|---|
| 1869 | Friedrich Miescher | Isolated an acidic, phosphorus-rich substance from the nuclei of white blood cells in pus on used bandages; he called it nuclein. | The nucleus contains a distinct chemical, later named nucleic acid. |
| 1928 | Frederick Griffith | Injected mice with Streptococcus pneumoniae. Heat-killed virulent S cells mixed with live harmless R cells killed the mice. | Something from dead S cells permanently changed R cells into S cells: the transforming principle. |
| 1944 | Oswald Avery, Colin MacLeod, Maclyn McCarty | Treated S-cell extract with enzymes that destroy protein, RNA or DNA, then tested whether it could still transform R cells. | Only destroying DNA stopped transformation, so DNA is the transforming principle. |
| 1950 | Erwin Chargaff | Measured the base composition of DNA from many species. | In every species %A = %T and %G = %C, but the proportion of A–T to G–C pairs differs between species (Chargaff's rules). |
| 1952 | Alfred Hershey, Martha Chase | Labelled bacteriophage T2 protein with or its DNA with and let the phages infect E. coli. | Phage DNA, not protein, enters the bacterium and directs the making of new phages. |
| 1951–1952 | Rosalind Franklin, Maurice Wilkins | X-ray diffraction of DNA fibres; Franklin and her student Raymond Gosling produced the image called Photo 51. | The X-shaped pattern showed a helix about 2 nm wide with regular repeats of 0.34 nm and 3.4 nm, and phosphates on the outside. |
| 1953 | James Watson, Francis Crick | Built a physical model that fitted Chargaff's rules and the X-ray data. | The double helix: two antiparallel strands, sugar-phosphate backbones outside, complementary base pairs inside. |
Griffith's experiment used two strains of the pneumonia bacterium. The S (smooth) strain has a polysaccharide capsule that hides it from the immune system and kills mice; the R (rough) strain has no capsule and is harmless. Live S cells killed mice, live R cells did not, and heat-killed S cells did not. But heat-killed S cells mixed with live R cells killed the mice, and Griffith recovered live S cells from their blood. The R cells had picked up something from the dead S cells, and their descendants stayed S, so the change was heritable. Griffith did not know what the substance was. Avery, MacLeod and McCarty purified it. When they treated the extract with a protease (destroys protein) or RNase (destroys RNA), transformation still happened. When they used DNase (destroys DNA), it stopped. Many scientists still doubted the result, arguing that a trace of protein might have contaminated the samples.
Hershey and Chase settled the question with a bacteriophage (a virus that infects bacteria), which is nothing more than a protein coat around a DNA core. They grew one batch of phages with radioactive sulfur, , and another with radioactive phosphorus, . The choice of isotopes was the key: sulfur is found in proteins (in the amino acids cysteine and methionine) but not in DNA, while phosphorus is in DNA's backbone but almost absent from protein. Each label therefore tagged only one molecule. After the phages infected E. coli, a kitchen blender sheared the empty phage coats off the cell surfaces, and a centrifuge spun the heavier bacteria into a pellet, leaving the light phage coats in the liquid supernatant. In the batch, the radioactivity stayed in the supernatant: protein never entered the cells. In the batch, the radioactivity was in the pellet, inside the bacteria, and some of it turned up in the next generation of phages. DNA was what the phage injected and passed on.
The structure of DNA
DNA is a polymer of nucleotides. Each nucleotide has three parts: a five-carbon sugar called deoxyribose, a phosphate group, and a nitrogenous base. The sugar's carbons are numbered 1' to 5' (read "one-prime" to "five-prime"). The base is attached to the 1' carbon, the phosphate to the 5' carbon, and the 3' carbon carries a hydroxyl (–OH) group. Nucleotides are joined by phosphodiester bonds running from the 3' carbon of one sugar, through a phosphate, to the 5' carbon of the next, forming a sugar-phosphate backbone. This gives every strand a direction: one end has a free phosphate on a 5' carbon (the 5' end), and the other has a free –OH on a 3' carbon (the 3' end). The two strands of the helix run in opposite directions, which is called antiparallel: beside a strand running 5'→3' is a partner running 3'→5'. The bases point inward and pair across the helix through hydrogen bonds, like the rungs of a twisted ladder. Franklin's X-ray images showed that the phosphates were on the outside and gave the helix's dimensions; Watson and Crick combined those measurements with Chargaff's rules and realized that A must pair with T, and G with C.
- Purines (adenine A and guanine G) have a double ring. Pyrimidines (cytosine C, thymine T, and uracil U in RNA) have a single ring. Memory aid: "pure as gold" for purines A and G; "CUT the pie" for pyrimidines C, U, T.
- Every base pair joins one purine to one pyrimidine, so every rung is the same length and the helix has a constant width of about 2 nm. Two purines would be too wide and two pyrimidines too narrow.
- A pairs with T through two hydrogen bonds; G pairs with C through three. DNA rich in G–C pairs therefore needs more heat to separate its strands.
- The two strands are complementary: the sequence of one strand fixes the sequence of the other. This is what makes copying possible.
- Each base pair adds 0.34 nm to the length, and one full turn contains about 10 base pairs (3.4 nm). The twist creates a major groove and a minor groove, where proteins can recognize base sequences without unwinding the helix.
Packaging: from double helix to chromosome
A diploid human cell holds about 6.4 billion base pairs (two sets of about 3.2 billion). At 0.34 nm per base pair, that is roughly 2 m of DNA in a nucleus about 6 µm across. Eukaryotes manage this with histones, small proteins rich in the positively charged amino acids lysine and arginine, which attract the negatively charged phosphate backbone. About 147 base pairs of DNA wrap nearly twice around a core of eight histones to form a nucleosome; in electron micrographs, nucleosomes joined by short linker DNA look like beads on a string. Another histone (H1) pulls the nucleosomes into a thicker fibre, which loops and folds further into chromatin. Loosely packed euchromatin can be transcribed, while densely packed heterochromatin is mostly silent. Before mitosis or meiosis, chromatin condenses further into the compact chromosomes visible under a light microscope.
- Usually one circular chromosome in the nucleoid region; no nucleus
- Often extra small circular DNA molecules called plasmids
- Small: E. coli has about 4.6 million base pairs and about 4300 genes
- Very little non-coding DNA; genes packed tightly, often grouped into operons
- DNA is supercoiled and held by proteins, but bacteria do not form nucleosomes
- No chromosome ends, so no telomeres
- Several linear chromosomes inside a nuclear envelope (46 in human body cells)
- Mitochondria (and chloroplasts in plants) also carry their own small circular DNA
- Large: about 3.2 billion base pairs per haploid set and about 20 000 protein-coding genes
- Mostly non-coding: only about 1–2% of human DNA codes for protein; introns and repeated sequences are common
- Wrapped around histones into nucleosomes and chromatin
- Chromosome ends capped by telomeres
Investigation: extracting DNA
| Step | What you do | Why it works |
|---|---|---|
| 1. Break up tissue | Mash strawberries, onion or split peas in a sealed bag. | Breaks cell walls and separates cells so the solutions can reach every cell. Strawberries are octoploid (eight sets of chromosomes), so they give a lot of DNA. |
| 2. Add detergent | Add an extraction buffer of dish detergent, table salt and water, and mix gently. | Detergent dissolves the phospholipid bilayers of the plasma membrane and nuclear envelope, the same way soap lifts grease, releasing the DNA. |
| 3. Salt (in the buffer) | The NaCl is already in the buffer. | ions shield the negative charges on the phosphate backbone, so DNA molecules stop repelling each other and can clump; salt also helps pull proteins such as histones off the DNA. |
| 4. Optional protease | Add a little meat tenderizer or pineapple juice. | These contain protease enzymes that digest histones and other proteins still bound to the DNA. |
| 5. Filter | Pour the mixture through cheesecloth or a coffee filter. | Removes cell-wall fragments and other debris; the DNA stays dissolved in the liquid that passes through. |
| 6. Cold ethanol | Slowly pour ice-cold ethanol (or isopropanol) down the side of the tube so it floats as a layer on top. | DNA dissolves in water but not in alcohol, so it precipitates as white threads where the layers meet. Cold lowers its solubility further and slows DNA-digesting enzymes (DNases). |
| 7. Spool | Wind the threads onto a glass rod or wooden skewer. | Long DNA molecules tangle together. What you collect is millions of DNA molecules (with some RNA and protein), not a single molecule. |