Restriction Enzyme maps
Maps of restriction enzyme cleavage sites are useful in planning not only cloning projects but also in gene identification, forensics, sequencing and mutagenesis projects.
Below is a description and examples of three methods used to organize restriction enzyme location on DNA fragments.
Estimating fragment sizes from RE map
There are both linear and circular maps.
We will start by estimating, from the maps, the fragment sizes that would be generated when cutting with particular enzyme of combinations of enzymes.
If you cut a linear DNA you will get one more fragment
than you have RE sites.
When cutting circular DNA, you get the same number of fragments as there
are RE sites.
Example, given the map below, find the sizes generated with the RE combinations:
| Restriction Enzyme | Fragment Sizes |
A |
600, 1000 |
B |
400, 800 |
C |
200, 1400 |
A + B |
200, 400, 600 |
A + C |
200, 400, 1000 |
B + C |
200, 400, 600 |
Try the circular map:
| Restriction Enzyme | Fragment Sizes |
A |
400, 1200 |
B |
1600 |
C |
200, 1400 |
A + B |
400, 600 |
A + C |
200, 400, 800 |
B + C |
200, 1200 |
Complete Digestion of unknown DNA
When mapping RE by complete digestion, often it is best to use information from both single and double digests.
Start with the simplest cuts first, for example the Bgl and Kpn single digests and compare them to the Bgl + Kpn double digest. Keep one of the single digest fixed (Bgl) and adjust the map fragments for the other single and double digest until both singles and the double digest cut sites align. Repeat this for each of the other single/double digest sets until the map of the RE sites are consistent throughout.
Complete digestion of DNA cloned into a known vector
When a fragment of DNA with an unknown restriciton map is cloned into a vector containing many flanking restriction enzyme sites, it is fairly easy to identify the location of most single cuts within the unknown portion.
For example, if a fragment cut with Cla I was inserted into a 2000 bp vector with the following multiple cloning site:
---- Eco RI --Pst I -- Cla I -- Bam HI -- Hind III ------
and subsequent cutting with each of the flanking enzymes created the following fragments, what RE map could you create?
Eco RI - 450, 2550
Pst I - 200, 2600
Bam HI - 3000
Hind III - 500, 2500
Answer:
-EcoRI-PstI-ClaI-(200)-PstI-(200)-PstI-(50)-EcoRI-(50)HindIII-(500)ClaI-BamHI-HindIII ---
Partial digest of end labeled DNA
Partial digest sites can be mapped from the labeled end. If the 5' end is labeled, then begin the map with the smallest fragments being located at the 5' end. If the 3' end is labeled then start the map with the largest fragments being located at the 5' end.