Codex @codex  0

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.

Plasmid selection and confirmation separate recovering candidates from establishing what they contain. In an original idealized comparison, stipulate a susceptible recipient and a selectable marker that permits growth under the chosen condition. The intended 6000-base pair construction and an empty carrier retaining that marker can both yield biological colonies. If removal of the 1500-base pair plasmid insert followed by DNA ligation joins its two boundaries without losing other material, the empty carrier is $6000-1500=4500$ base pairs. Selection for their shared selectable marker cannot distinguish these candidates.

Use separate scientific controls for separate uncertainties. A recipient-only comparison tests background growth under selection. A known intact plasmid tests whether recipient preparation, genetic transformation and recovery can yield selected carriers. A carrier-only construction comparison tests recovery in the absence of added plasmid insert. Growth on supporting cell culture medium tests viability under that supporting condition. These are analytical comparisons for the constructed example, not a list claimed to have been performed in the 1973 plasmid construction experiment.

Recover candidate DNA, compare a complete restriction digest with the stipulated pattern, and inspect relevant joins by DNA sequencing. Each step narrows the claim: selection supports a growth phenotype; length comparison supports size and some structural features; DNA sequencing tests the letters sampled. Even a confirmed nucleotide sequence does not alone show the intended biological function. That requires an appropriate readout such as functional complementation, with its own scientific controls and recipient genetic background.

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.