Sanger sequencing

Meaning: Also called the chain-termination method. A single-stranded DNA template, a primer, DNA polymerase, all four dNTPs and a small amount of ddNTPs are combined. A ddNTP has no 3′-OH, so once it is added, the next nucleotide cannot be joined and the strand ends. Termination happens at random positions, so the mixture contains fragments that differ in length by one nucleotide, each ending in a known base. Fragments are separated by size with gel or capillary electrophoresis. In automated versions each ddNTP carries a different fluorescent dye.

On the MCAT: A passage shows a gel with four lanes (A, T, G, C), or a colored peak trace. You read from the shortest fragment to the longest, which gives the new strand 5′ → 3′. Small fragments travel farthest toward the positive electrode, because DNA is negatively charged. You may also be asked to predict the result of a change. Too much ddNTP gives mostly short fragments. No ddNTP gives only full-length products, so no ladder. A ddNTP that is missing means one base never appears as a terminating band.

Common confusion: The bands spell the synthesized strand, which is complementary to the template, not the template itself. Complement it to get the template sequence. Also, the shortest fragment is the 5′ end, so read from the bottom of the gel upward.