Biology · Introductory biology · Concept
Restriction digests and gels
Connect DNA cut positions to fragment lengths, then interpret the pattern those fragments make on a teaching gel.
Separate sequence recognition from the geometry of cutting
A restriction enzyme recognizes a DNA sequence and cuts at a defined position relative to it. If you already know the cut positions, you can study fragment lengths without finding the recognition sequences. This lesson uses that measured-length model; it does not identify an enzyme from positions alone.
Choose linear or circular DNA first
Linear DNA has two ends. Each distinct internal cut adds one fragment, so two cuts make three fragments. Circular DNA has no free ends until it is cut: one cut opens it into one linear fragment, and two distinct cuts make two fragments.
What about an uncut circle?
An uncut circular molecule is still one molecule, not zero fragments. Its migration may depend on conformation. The simple size-based teaching gel is best used for interpreting the linear fragments produced by the modeled digest.
Put the cuts in order and subtract neighboring positions
For a 6000 bp linear molecule with cuts at 1000 and 3500 bp, include boundaries at 0 and 6000. The distances between neighboring boundaries are the fragment lengths. Read the diagram from left to right rather than subtracting each cut from the total length.
Use total length as a check
A complete digest rearranges the original DNA into pieces without changing its total number of base pairs. The fragment lengths should therefore sum to the molecule’s original length. If they do not, look for a missing end segment, a duplicate cut, or the wrong topology.
Distinguish fragments from band positions
In a size-based agarose-gel model, shorter linear DNA fragments generally migrate farther. Fragments of the same length can overlap at one position. Three fragments of 1000, 2500 and 2500 bp therefore give two distinct size positions, not three.
What can band brightness tell us?
Real staining intensity can depend on DNA mass and experimental conditions. The current teaching gel does not provide a quantitative intensity measurement, so do not infer an exact amount from its brightness. A shared band position also does not prove that two fragments have the same sequence.
Carry the assumptions into the gel
The predicted fragments assume every configured site is cut completely. A partial digest can contain additional sizes. A ladder supplies known sizes for comparison; choose an appropriate ladder in Gel rather than treating an unlabeled lane as a calibration.
Common mistakes
- Forgetting the fragment between the final cut and the end of linear DNA.
- Using the same fragment-count rule for linear and circular DNA.
- Equating the number of visible band positions with the number of fragments.
Work through an example
A linear 6000 bp molecule has cuts at 1000 and 3500 bp. Find its fragments and predict the visible band positions.
Predict a digest and its gel pattern →Sources and scope
Authored study material. Tool results depend on the stated inputs and model assumptions.