Restriction Enzymes

(Pre-lab for Exercise 3)

Goals:
  1. understand agarose gel electrophoresis as explained in the lab manual. Learn how to prepare and load an agarose gel.
  2. learn how restriction enzymes function and how to set up a restriction digest.

 
Restriction Enzymes
   Cutting DNA by using restriction enzymes is one of the most common Molecular Biology techniques. The availability of pure restriction enzymes was one of the first major advances in the new science of Molecular Biology. These enzymes occur naturally in bacteria. Restriction enzymes are used to protect the bacteria from invading foreign DNA such as bacterial viruses (bacteriophage). They work by cutting the DNA at a specific nucleotide sequence, the recognition sequence of the enzyme. Any time this sequence appears in DNA it will be cleaved by the enzyme, whether it is viral or frog or human DNA. This is why restriction enzymes are so critical to molecular biology; they are very specific, cutting only at their unique recognition sequence, but they are also general because they will cut any DNA having this sequence. This has made cloning and other DNA manipulations possible.
   The recognition sequence contains an even number of nucleotides. Many enzymes recognize a 6-base pair (bp) sequence. The recognition sequence is a palindrome. An enzyme recognizes its specific palindromic sequence, sits down on the DNA at that spot, and cleaves the DNA. Both strands of the DNA helix are cleaved, but the enzyme can leave either "blunt" or "sticky" ends. If the enzyme cuts unevenly, leaving either a 3' or a 5' overhang on the helix, this is a sticky end. If the enzyme cuts the helix at the same point on both strands, this leaves blunt ends.

 

    Restriction enzymes are named for the bacteria where they were isolated, using the following method: the name of the enzyme is taken from the first letter of the bacterial genus and the first two letters of the bacterial species. Thus the first three letters of the restriction enzyme name come from a formal genus and species designation. They must therefore be italicized or underlined, just like you would do for the bacterial species itself. The enzyme name may have additional numbers or letters. These may designate a particular strain, isolate, or plasmid. These numbers or letters should not be italicized or underlined. Below are examples of the enzymes we will use in lab:
 
 
enzyme name
bacterial species
5' -> 3' recognition sequence
EcoR I
 Escherichia coli RY 13 
G'AA TT  C
BamH I
 Bacillus amyloliquefaciens H 
G'GA TC C
Hind III 
  Haemophilus influenzae Rd 
 A'AG CT T

Here is an example of an enzyme that leaves a blunt ended cut:
SnaB I 
 Sphaerotilus natans 
      TAC' GTA 

    DNA fragments with blunt ends or those with sticky ends can be rejoined. However, there is a major difference between a fragment with a blunt end and one with a sticky end. Any blunt-ended fragment is compatible with any other blunt-ended fragment to form a new DNA molecule. But a fragment with a stick end can only join with another fragment that has the complimentary "sticky" nucleotides. Thus if you cut DNA with SnaBI, the ends can be joined to any other piece of DNA that has been cleaved to leave a blunt end. But DNA cut with EcoRI is only compatible with another piece that has also been cut with EcoRI, because the 4-base overhang can line up and hydrogen bond. (try to draw out the cutting and rejoining of two EcoRI fragments)
 
 

    All enzymes are measured in units of activity. The activity varies from enzyme to enzyme, depending on its function. The activity of restriction enzymes is the ability to cleave DNA. Thus the definition of one Unit (U) of restriction enzyme activity is:
 

The amount of restriction enzyme needed to completely digest (cleave) one microgram (ug) of substrate DNA (often Lambda DNA) in one hour at the optimal temperature of the enzyme (usually 37'C) in a 50-ul reaction volume.
You should know this definition. You should also understand what this really means when it comes time to set up a restriction digest. The number of units of activity will help you determine how much of the enzyme to use in a restriction digest. You need to use enough enzyme to cut the DNA to completion. However, too great of an excess may be detrimental to the digest. Also, these enzymes are expensive. Using more than we need wastes money.
    Each enzyme has certain requirements for optimal activity, including the proper restriction enzyme buffer and incubation at the correct temperature. The enzyme's manufacturer will provide buffer and instructions for optimal digest activity. Nearly all of our enzymes come from the Promega Company. Thus we use Promega buffers and the Promega catalog to help determine the best digest conditions.

 

Setting up a Restriction Digest

    Since the restriction digestion of DNA is one of the most common techniques of Molecular Biology, it is important to understand how digests are carried out. All restriction digests contain the following components:
 

   The question is, how do you determine the quantity of each of these components? Because restriction digests are done for different outcomes, there are no absolute hard and fast rules. However, there are general guidelines to follow whenever you set up a digest.
 
 
1. DNA

    You must determine the amount of DNA to be cut, and the volume of DNA stock solution that is required. Look on p. 362 of the lab manual, where the restriction digests are set up. The authors use 4 ul of Lambda DNA. This DNA has a concentration of 0.1 ug/ul. So how many ug of DNA are being cut in this digest?_______
 

2. Restriction enzyme

    Remember the definition of a Unit (U) of restriction enzyme activity. You must determine how many Units are needed to cut the DNA. Then you have to decide what volume of enzyme will contain sufficient Units of activity to accomplish this task. Most of the Promega enzymes that we use have a concentration of 10 U/ul. Knowing the definition for a Unit of activity, how many micrograms of DNA can be cut with one microliter of a Promega enzyme?_____  How many Units would be needed to cut the 4 ul of Lambda DNA in Exercise 3?______ These values will be difficult to understand at first. However, these are the types of calculations that you need in order to determine how much enzyme to use and they will become easier with practice.
 

There are two general guidelines for determining the amount of enzyme:

(a) Use at least the minimum number of Units necessary to cut the DNA. Most people use more Units than absolutely necessary. This speeds up the time needed and helps insure a complete digest.

(b) Try to keep the total volume of restriction enzyme in the digest to 10% or less of the total digest volume. Thus if you have a 30 ul digest, don't use more than 3 ul total of enzyme.

Finally, always add the enzyme last. Even though I have listed it as the second component above, the enzyme is always last so it can be in the optimal reaction conditions and start to work immediately.
 
 
3. Buffer

    Each enzyme has its optimal buffer. The first task is to make sure you use the right buffer. Then you must determine the volume of buffer to add to the digest. Restriction enzyme buffers are often sent as a 10X stock concentration. We always use a 1X working concentration in lab. This means that the stock is ten times more concentrated than the working solution. Therefore, the stock solution must be diluted 1:10 in the restriction digest. This is not as difficult as it sounds. It is simply a matter of setting up a (C1)(V1) = (C2)(V2) proportion. If your final restriction digest volume is 30 ul, then you use 3 ul of the 10X stock buffer.
 

4. Water
 

    Finally, water is added to whatever volume is not taken up by DNA, buffer, or enzyme. Often, when you actually set up a digest, the water is added first because it is the largest volume. Then it is easy to mix the buffer and the DNA into the water.
 

Here is a sample restriction digest:

Cleave 2 ug of DNA with EcoRI in a 40 ul reaction volume
 

STOCK DIGEST
DNA 0.5 ug/ul 4 ul
buffer H 10x 4 ul
EcoRI 8 Units/ul 0.5 ul
water   31.5 ul
TOTAL   40 ul


The lab manual has useful information on the following topics:


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