Restriction Enzymes

- Also: Restriction Endonucleases

- Cuts double-stranded or single-stranded DNA at specific recognition nucleotide sequences known
as restriction sites.
- Thought to have evolved to provide a defense mechanism against invading viruses.
- Types: Type I, Type II, Type III, and Type IV
o They differ in
 Recognition sequence
 Subunit composition
 Cleavage position
 Cofactor requirements
- Examples (few among the many):
o HindIII
o EcoRI
o BamHI
o TaqI
o PstI

Restriction Mapping
- Mapping of known restriction sites within a sequence of DNA (plasmid).
- Molecular biology, restriction maps are used as a reference to engineer plasmids or other
relatively short pieces of DNA, and sometimes for genomic DNA.
- Goal: be able to find the order of the fragments, knowing just the fragment lengths.

Sample problem:

Plasmid pRIT451 was cut with Sma I, BgI II, and Ava I. From the data below, determine the map.

Sma I 5.9 4.3

Bgl II 8.2 2.0
Ava I 5.3 4.9
Sma I + Bgl II 5.4 2.8 1.5 0.5
Sma I + Ava I 3.3 2.6 2.3 2.0
Ava I + Bgl II 5.3 2.1 2.0 0.8
Solution:

1. We first analyze the given data, noting the total size of the whole plasmid (10.2 kb), how many
fragments were generated for every single digests (2 fragments), and double digests (4
fragments).

2. If the plasmid pRIT451 is cut with SmaI, AvaI, and BglII separately or singly, two fragments
would be generated for every restriction enzyme. Since it is easy to map the plasmids if only one
 restriction enzyme is used, we will draw each one to have an idea visually on how the fragments
would look like and use this as a reference to generate the maps for the double digests.
SmaI SmaI SmaI
5.9 4.3

AvaI AvaI AvaI

5.3 4.9

BglII BglII BglII

8.2 2.0

3. We will now begin by arbitrarily choosing BglII, but we could have just easily chosen to do AvaI
or SmaI first, it makes no difference, actually. We first look at BglII + SmaI. But we could have
also chosen to do BglII + AvaI first; it does not really matter because the final outcomes will still
be the same. You can try to do BglII + AvaI first later, if you don’t believe me. Hehe 
 It helps to think of the double digest conceptually as a two-step process in which the
plasmid is first cut by BglII into the two reference fragments, and then SmaI cuts the
reference fragments late on.

4. In the BglII + SmaI double digest, we generate 4 fragments. Combinations of the fragments will
sum up to the reference fragments. Here, since we have 2 fragments for the BglII single digest,
we have to find the combinations of the 4 fragments from the BglII + SmaI double digest that will
be equal to the reference fragments drawn earlier.

BglII BglII BglII

8.2 2.0

We observe that 5.4 + 2.8 = 8.2 and 1.5 + 0.5 = 2.0. Thus, we can organize the fragments as such:
B S B S B
5.4 2.8 1.5 0.5

5. This is not the final positioning of the fragments. This is just an idea on where the fragments may
be placed. In order to be sure, we draw all the possible positions of the fragments relative to each
other, taking into mind the possible combinations of the 4 fragments based on the 2 reference
fragments.
Orientation A:
B S B S B
5.4 2.8 1.5 0.5

Orientation B:
B S B S B
5.4 2.8 0.5 1.5

Note that we did not change the position of the fragments in the larger reference fragment since if we
change it as well; we are just making it the same as the positioning in the Orientation A.
 6. We must next determine which orientation is correct. In orientation A, the two SmaI sites are far
apart (distal), and in B, the SmaI sites are closer (proximal). Clearly, an SmaI single digest will
produce the fragments in different orders and we can verify by determining expected fragment
sizes for each orientation and compare our expected values with the values observed:

Orientation A: Orientation B: Observation (SmaI):

2.8 + 1.5 = 4.3 2.8 + 0.5 = 6.9 5.9 + 4.3 = 10.2
5.4 + 0.5 = 5.9_ 1.5 + 5.4 = 3.3_
10.2 10.2
By comparing our expected and observed values, we find that orientation A is the correct orientation.

B S B S B
5.4 2.8 1.5 0.5

7. Using the same logic, we can determine the correct orientation of the AvaI sites, with respect to
our reference fragments as is below: (kayo nalang magcompute para macheck hoho)
B A A B B
2.1 5.3 0.8 2.0

8. To complete the map, we must now superimpose the SmaI and AvaI maps.

B S B S B
5.4 2.8 1.5 0.5

+
B A A B B
2.1 5.3 0.8 2.0

B A S A B S B
= 2.1 3.3 2.0 0.8 1.5 0.5

Check the fragment lengths dun sa given data, makikita nyo na magkocoincide sila
especially kung tama yung order at positioning. Use same principle as #6 pero this time,
between two different sites.

9. Lastly, we map. It doesn’t really matter where you are going to start or what fragment you are
going to start with. As long as
AvaI
the fragments are of the 3.3
correct order, that
SmaI
would be fine.
2.1
pRIT451
10.2 kb 2.0
BglII
0.5

SmaI 0.8
AvaI
1.5
BglII
A few reminders:
 Guys, this is a very tedious and laborious activity. It requires patience and of course, practice.
(Took me days before I was able to solve one problem in 5 minutes huhu.)
 It really is a trial-and-error kind of problem solving. So if you get lost, just start over again
but do not make the same mistakes again. Charrr. XD
 Problems can be found in some microbial genetics books and of course in the internet. So if
you want to practice, just google in “Restriction mapping problems”, and if you don’t have
time to surf the net, you can play with the values to give different data, so that you can
practice more.
 Choosing where to place the fragments really is arbitrary. It’s up to you where you place
fragments and how you position them in the map. As long as you still use the principles in the
steps mentioned above.
 Go classmates! Kaya natin to!!! Hehe.