= Sanger ladder reading
{c}
Consider an original teaching template whose next eight positions, encountered after the <sequencing primer>, are
$$
3'\text{-}\mathrm{TGCAATCG}\text{-}5'.
$$
The <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 extension
$$
5'\text{-}\mathrm{ACGTTAGC}\text{-}3'.
$$
These eight letters are added material, not the entire primer-containing <DNA> <molecule>.
\Image[/sanger-four-lane-ladder-reading.png]
{title=Read an original four-lane <Sanger sequencing> ladder from the shortest fragment upward to recover $5'\text{-}\mathrm{ACGTTAGC}\text{-}3'$}
{description=Invented ideal endpoints: A at extension positions 1 and 6; C at 2 and 8; G at 3 and 7; T at 4 and 5. Every fragment contains the same <sequencing primer> of length $p$; the shortest drawn band is $p+1$ <nucleotides>, not a one-<nucleotide> whole strand. The gel is a schematic of <denaturing polyacrylamide gel electrophoresis>; band spacing, widths and darkness are illustrative, with no measured migration, abundance or experimental photograph. The right panel shows the complete added prefix represented by each endpoint.}
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>.
|| Added position $n$
|| Reaction lane
|| Added prefix, $5'$ to $3'$
|| Whole-fragment length
| 1
| A
| A
| $p+1$
| 2
| C
| AC
| $p+2$
| 3
| G
| ACG
| $p+3$
| 4
| T
| ACGT
| $p+4$
| 5
| T
| ACGTT
| $p+5$
| 6
| A
| ACGTTA
| $p+6$
| 7
| G
| ACGTTAG
| $p+7$
| 8
| C
| ACGTTAGC
| $p+8$
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 $5'$-to-$3'$ 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:
$$
\begin{aligned}
\text{new extension:}&\quad 5'\text{-}\mathrm{ACGTTAGC}\text{-}3',\\
\text{aligned template:}&\quad 3'\text{-}\mathrm{TGCAATCG}\text{-}5'.
\end{aligned}
$$
If the requested answer is that template written $5'$ to $3'$, reverse the aligned template to obtain $5'\text{-}\mathrm{GCTAACGT}\text{-}3'$, 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 $3'$-to-$5'$ direction, but not in the usual reporting convention. Specify the strand and its direction before transforming the answer.
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