What is the difference between single digestion and double digestion when discussing restriction enzymes?

NOTE: Endonucleases should be read in place of REs (Restriction Enzymes) in this response.

NOTE2: If you're not familiar with DNA fingerprinting, check out these two resources.

For example, Hin DIII will only make a cut in the sequence: This demonstrates how highly specific REs are for the DNA sequence they splice, which is almost always a predetermined Palindromic sequence.

This palindorme has six bases. I won't go into statistical analysis, but it's safe to assume that this will lead to fewer cuts than, say, the activity of Sau3A:

Utilizing Sau3A to digest (splice or cut) a human's total nuclear DNA for "fingerprinting" may not yield the intended outcome due to the excessive number of tiny fragments that result in an incomprehensible "smear" on your Electrophoresis Gel slab.

You would like more certainty, but cutting with Hin DIII will produce a few large bands that can be telling.

But here's where things get tricky: while DNA is relatively robust and inert, REs are not. Because they were taken from distinct organisms (bacteria), each RE will have its own ideal environment within the host cell, which means each RE will have its own ideal buffer mixture (pH is one thing to consider, among other things). And that's where the trouble starts.

While most REs have been found to be fairly happy in the laboratory using TRIS-based buffers (Tris-HydroxyMethyl AminoMethane), such as TBE (Tris/Borate/EDTA), each RE will require adjustment of the selected buffer for optimal activity. Temperature is one of the variables that can affect this.

Therefore, if the two REs are compatible buffer-wise, you could perform a double digest in one step; however, based on how well your two REs work together, you might need to perform the double digest in two stages: halt the first digest's reaction (heat shock), purify the DNA digest, and then perform the second digest.

However, as of this writing, more than 3000 REs have been found, and more than 600 of them are offered for sale, so I won't be providing you with a comprehensive list of possible combinations.

A list of compatible REs and their buffers for double digestion may be provided by Bethesda Labs (USA), New England Biolabs (USA), or Boehringer Ingelheim (Germany), from whom we obtained the majority of our REs in the 1980s.

Single digestion involves cutting DNA with one restriction enzyme, while double digestion involves cutting DNA with two different restriction enzymes.