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DNA sequencing determines the order of nucleotide bases in DNA. Sequence data can test molecular rearrangements, as in the functional plastid-to-nucleus gene transfer experiment, or support phylogenetics after a suitable sequence alignment.

An ordered nucleotide sequence answers a different question from fragment size. The eight-nucleotide strands ACGTTAGC and AACCGGTT have equal lengths and identical counts of adenine, cytosine, guanine and thymine, yet different orders. A length-compatible gel band therefore cannot identify either strand by itself. Sanger sequencing makes length informative by deliberately coupling each stopping position to the identity of its last added nucleotide.

That coupling is the beautiful experimental idea: convert an invisible order into a collection of ordered endpoints. Sanger ladder reading recovers the newly synthesized strand; base pairing and the reverse complement then connect that readout to the original template. The distinction also matters when checking a mutation: a reliable local nucleotide sequence supports a particular molecular change, while its effect on a phenotype requires additional evidence.

Consider an original teaching template whose next eight positions, encountered after the sequencing primer, areThe sequencing primer binds an upstream region omitted from this display; the template also continues beyond the eight positions shown. DNA polymerase encounters the displayed template from left to right. Applying base pairing at each successive position gives the extensionThese eight letters are added material, not the entire primer-containing DNA molecule.

Here is the full endpoint accounting. A terminal letter denotes an incorporated terminating nucleotide; all preceding letters of that extension were supplied by ordinary deoxynucleoside triphosphates.

The shortest fragments migrate farthest, placing position 1 at the bottom. Reading upward, the lane labels spell A, C, G, T, T, A, G, C: the new strand in its $5^{\prime }$-to-$3^{\prime }$ direction. The adjacent T bands at positions 4 and 5 encode two separate additions. A single unresolved T patch would leave that count uncertain. In the A lane, the position-6 fragment passed position 1 using ordinary dATP and stopped at position 6 using ddATP; it does not skip the intervening nucleotides.

The common sequencing primer is an offset. For primer length $p$, endpoint $n$ has length $p+n$ nucleotides. With an illustrative $p=20$, the drawn fragments would contain 21 through 28 nucleotides, and adjacent endpoints still differ by one. Subtracting the common $p$ preserves their order because $(p+n+1)-(p+n)=1$. This supplies a short justification for Sanger ladder reading: nested fragments share a start, length orders their endpoints, and lane membership supplies each endpoint's letter. A missing early band cannot be relabeled position 1 without independent evidence about the starting point.

To report the original template region, first complement the readout in aligned orientation:If the requested answer is that template written $5^{\prime }$ to $3^{\prime }$, reverse the aligned template to obtain $5^{\prime }\text{-}\mathrm{GCTAACGT}\text{-}{3}^{\prime }$, the reverse complement of the readout. Reversing the readout alone would give CGATTGCA and would be wrong. Complementing without reversing gives the correct template letters in its displayed $3^{\prime }$-to-$5^{\prime }$ direction, but not in the usual reporting convention. Specify the strand and its direction before transforming the answer.