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EcoRI is a restriction enzyme whose usual recognition sequence is GAATTC. Staggered cleavage produces complementary single-stranded ends.

A plasmid feature map records the positions of specified DNA features. Construct an original circular example of total length $L=6000$ base pairs. Coordinates run clockwise from a chosen zero, and $0$ and $6000$ label the same boundary. Positions here denote boundaries between base pairs, so the interval from 1000 to 2500 contains $2500-1000=1500$ base pairs. This convention avoids counting both endpoints as additional material.

The origin of replication is assigned to 200–700, the selectable marker to 3200–4000 and the plasmid insert to 1000–2500. Other positions are unspecified DNA, not empty gaps. Their lengths account for the entire circle:The first three terms are the marked regions; the last is all remaining material. Choosing a coordinate zero does not add a physical break. Rotating every coordinate by the same amount changes the drawing's labels without changing any distance around the plasmid.

A gene promoter must function in the recipient for the intended gene expression; copying a plasmid alone does not establish gene expression.^[ref] The map therefore supports three separate questions: can the carrier persist, can carriers be recovered, and can the selected DNA perform the proposed task? The galactose induction comparison illustrates the third question in an actual yeast experiment.

Plasmid restriction mapping compares fragment lengths after a restriction digest with the pattern predicted by specified cut positions. Use the original 6000-base pair plasmid feature map. Abstract restriction enzyme A cuts once, at 1000. Abstract restriction enzyme B cuts twice, at 2500 and 4500. Assume complete cutting of identical circular copies at exactly those sites, with no loss of DNA. These assumptions define a length-accounting exercise; the labels A and B do not stand for particular commercial enzymes.

For the combined restriction digest, successive clockwise distances areand the final fragment crosses the chosen zero:Their sum is $1500+2000+2500=6000$. The single A cut opens the circle into one full-length linear fragment. It does not produce pieces of lengths 1000 and 5000, because coordinate zero was never an end.

For contrast, stipulate a different starting substrate: a linear 6000-base pair DNA fragment with physical ends at 0 and 6000 and the same three internal cut coordinates. Its complete restriction digest givesbase pairs, summing again to 6000. There are four fragments, including two of equal length. Even an ideal length-resolving comparison would show only three distinct sizes if both 1500-base pair fragments occupy the same gel band. Fragment number and visible gel band number need not agree.

The general counting argument is short. For $k\ge 1$ distinct complete cuts of a circle, each fragment runs from one cut to the next, so there are $k$ fragments. For a linear substrate with $k$ distinct internal cuts, its two pre-existing ends give $k+1$ intervals. Summing successive distances counts every original base pair once in either case. The rule assumes internal double-strand cuts and does not treat an uncut circle as a zero-length product.

In practice, compare released linear fragments with an appropriate DNA ladder using agarose gel electrophoresis. The original uncut circular DNA need not migrate like a linear fragment of equal length; the existing agarose gel electrophoresis discussion explains that shape-dependent limitation. Partial cutting also changes the expected pattern. In this example, a circular copy cut at 1000 and 2500 but not 4500 yields lengths 1500 and 4500. A 4500-base pair product would therefore be compatible with that missed cut rather than uniquely identifying a different construction.

Matching lengths constrain an arrangement without determining every nucleotide sequence. For example, any change of letters preserving all three cut positions leaves this predicted pattern unchanged. Even the order of fragments can be ambiguous when only their unordered lengths are observed. DNA sequencing across both joins and through the plasmid insert tests additional claims that this length model cannot decide.

A restriction digest is cleavage of DNA using a restriction enzyme. Complete cutting at all specified sites and partial cutting can give different fragment patterns.

In 1977, Frederick Sanger, S. Nicklen and Alan Coulson tested chain termination on Phi X 174 DNA. They isolated restriction enzyme fragments as primers, heated them with template to $100^{\circ }\mathrm{C}$ for three minutes and annealed at $67^{\circ }\mathrm{C}$ for thirty minutes. Four extension mixtures used the Klenow fragment of DNA polymerase and radioactive dATP. Their Figure 1 used ddGTP, ddATP, ddTTP and araCTP, an alternative terminating substrate; Figure 2 used all four dideoxy types. This distinction matters when relating the original experiments to an all-ddNTP teaching model.^[ref]

Figure 1 used a 12% acrylamide gel at $40\mathrm{mA}$ for fourteen hours, read by autoradiography. Omitting the unlabeled dATP chase caused unwanted A stops across samples: a chemistry scientific control. Ordered bands supported local nucleotide sequences; comparisons could revise provisional assignments. Weak T bands, unresolved long runs and structure-dependent migration limited interpretation. The paper's observations are evidence from particular preparations, not a guarantee that every endpoint is resolved.^[ref]