DNA Replication

Meselson–Stahl experiment

Grew bacteria in heavy (¹⁵N) then light (¹⁴N) nitrogen and tracked DNA density by centrifugation across generations. Results ruled out conservative and dispersive replication and confirmed semi-conservative replication (each new helix has one parental and one new strand).

Three replication models

Conservative: one helix all-old, one all-new. Semi-conservative: each helix has one old + one new strand (correct model). Dispersive: both helices are mixed patches of old/new.

Kornberg's experiment

Showed a cell-free (in vitro) extract could synthesize DNA, identifying DNA polymerase as the enzyme responsible.

dNTPs

Deoxynucleoside triphosphates are the substrates for DNA synthesis; as each is added, its extra phosphates are cleaved off (energetically favorable), driving the reaction forward.

Replication machinery/process

Initiator proteins bind the origin → helicase unwinds DNA → topoisomerase relieves supercoiling ahead of the fork → primase lays down an RNA primer → single-strand binding (SSB) proteins keep strands separated → DNA polymerase extends 5'→3'.

Leading vs. lagging strand

Leading strand: synthesized continuously toward the fork. Lagging strand: synthesized discontinuously (away from the fork) as Okazaki fragments, later joined by DNA ligase.

Bidirectional replication / multiple origins

Replication proceeds in both directions from an origin. Bacteria have one origin (but can start new rounds before finishing); eukaryotes have many origins to replicate large genomes quickly.

Processivity

The ability of a polymerase to stay attached to the template and synthesize long stretches without falling off.

End-replication problem

The lagging strand can't complete synthesis to the very end of a linear chromosome once the terminal RNA primer is removed (no 3'-OH to extend) — chromosomes would shorten every division.

Telomeres & telomerase

Telomeres are repetitive protective end-sequences (discovered by Blackburn & McClintock). Telomerase (RNA-dependent DNA polymerase; discovered by Greider & Blackburn) carries its own RNA template and extends the 3' end using reverse transcription, solving the end-replication problem. Cells stop dividing once telomeres get too short.

DNA sequencing (Sanger)

Uses dideoxynucleotides (ddNTPs, lack the 3'-OH) that terminate strand extension when incorporated. Original method used one base per reaction + gel electrophoresis; modern method uses fluorescently labeled ddNTPs (one color per base) read by capillary electrophoresis in a single reaction.

Next-generation sequencing (NGS)

Cheap, high-throughput sequencing now used clinically; can detect single base-pair mutations.

PCR (Polymerase Chain Reaction)

In vitro DNA amplification. 1) Denature (heat separates strands); 2) Anneal (cool, primers bind); 3) Extend (Taq polymerase, heat-stable, synthesizes new strand). Ingredients: template DNA, primers, dNTPs, Taq polymerase, buffer.