DNA sequencing

Meaning: Sanger (chain-termination) sequencing copies a single-stranded template using a primer, DNA polymerase, the four normal dNTPs, and a small amount of fluorescently labeled dideoxynucleotides (ddNTPs). A ddNTP has no 3′-OH, so once it is added, polymerase cannot attach the next nucleotide and the chain stops. Termination happens at random positions, so the fragments collectively end at each position along the template. Electrophoresis separates the fragments by size, with the shortest travelling farthest. Reading from shortest to longest gives the new strand 5′→3′. Newer high-throughput methods read many short fragments in parallel.

On the MCAT: A passage shows a gel or colored chromatogram and asks for the sequence, usually of the template or its complement. You may also predict the effect of changing conditions: more ddNTP gives shorter fragments, and no ddNTP means no termination. Questions may ask why a primer is needed (polymerase extends only from an existing 3′ end).

Common confusion: Students read the bands as the template sequence. The ladder spells the newly synthesized strand, so the template is its complement and runs antiparallel to it. Another mix-up is with PCR, which also copies DNA but uses normal dNTPs to amplify DNA rather than to read its sequence.