Restriction Enzyme Analysis of DNA- Student Handout

How to set up a restriction enzyme reaction

Restriction enzymes (or restriction endonucleases) cleave DNA in a very specific fashion. Type II restriction enzymes, most commonly used for DNA analysis and genetic engineering, each have a unique nucleotide sequence at which it cuts a DNA molecule. A particular restriction enzyme will cleave DNA at that recognition sequence and nowhere else. The recognition sequence is often a six base pair palindromic sequence (the top DNA strand from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or even eight base pair sequences.

Restriction enzymes can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or blunt end. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called cohesive ends, as these single-stranded overhangs could potentially come together again through complementary base-pairing.

A common use for restriction enzymes is to generate a "fingerprint" of a particular DNA molecule. Because of the sequence specificity of restriction enzymes, these enzymes can cut DNA into discrete fragments which can be resolved by gel electrophoresis. This pattern of DNA fragments generates a "DNA fingerprint," and each DNA molecule has its own fingerprint. Other restriction enzymes can be used to further characterize a particular DNA molecule. The location of these restriction enzyme cleavage sites on the DNA molecule can be compiled to create a restriction enzyme map. These maps are very useful for identifying and characterizing a particular DNA plasmid or region.

In this laboratory, you will use restriction enzymes to generate DNA profiles for bacteriophage lambda, a bacterial virus.

How to set up a restriction enzyme reaction:

A restriction enzyme reaction contains the DNA to be analyzed, a restriction enzyme, and a restriction enzyme buffer mix. Each restriction enzyme prefers a particular restriction enzyme buffer mix. This buffer mix, commonly supplied at a 10X concentration, contains a buffering agent (usually Tris) to maintain constant pH, salt (usually NaCl or KCl) to provide the correct ionic strength for the digest, and Mg++ (from MgCl2) as a necessary cofactor for enzyme activity.

Commercially available restriction enzymes usually have activities at 10-20 units/ul. A "unit" is usually defined as the amount of enzyme needed to digest 1 ug of bacterial virus lambda DNA in 1 hour in a 50 ul reaction. We generally use 10-20 units (1 ul) of restriction enzyme per reaction. This is usually far more than needed, but this excess assures that complete digestion will occur. Digestion times are 1.5 hours, but can be lengthened. After the reactions are completed, the samples can be stored in the refrigerator until ready to use. Loading dye is added to the DNA digests, and the samples loaded onto a gel.

In this laboratory you will treat bacteriophage lambda DNA with either the restriction enzyme Hind III or Eco RI. This restriction enzyme digest will create a pattern of specific DNA fragments that serves as a "DNA ladder. " Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated.

Materials and Equipment

37^oC incubator or water bath
Electrophoresis units and power supplies
0.8% agarose in 1X TAE (melted)
1X TAE for electrophoresis units
Methylene blue solution
Staining trays
Light boxes

For each group of four

Lambda DNA (0.5 ug/ul)
Deionized or distilled H2O
10X React 2 restriction enzyme buffer
10X React 3 restriction enzyme buffer
Eco RI (10 units/ ul)
Hind III (10 units/ul)
Microcentrifuge tubes
Micropipets and tips

Perform the following steps:

Day 1: Set up the restriction enzyme reaction

1. Each member of the team will do a restriction enzyme digestion of bacteriophage lambda DNA. Set up the restriction enzyme digest as shown below (listed in order of addition):
   
   

   Lambda DNA (0.5 ug/ul)	6ul
   Deionized or distilled H2O	10 ul
   10X React 2(Hind III) or 10X React 3 (EcoRI)	2 ul
   Hind III or Eco RI	2 ul
   Total volume	20ul

   
   
   
2. Pool the reagents at the bottom of the tube. The tubes can be tapped to collect the fluids at the bottom.
   
   
3. Incubate reaction tubes for at least 1.5 hours at 37 C (in a water bath or incubator). Store reactions in the refrigerator until next class (this will be done for you).
   
   

Day 2: Agarose gel electrophoresis

1. Pool the liquid from the restriction enzyme reaction at the bottom of the tube. Add 4 ul 6X loading dye to each reaction tube. Be sure the loading dye mixes with the DNA sample at the bottom of the tube.
   
   
2. Record your electrophoresis unit number on your data sheet. Set up the electrophoresis unit by inserting the black casting dams and placing a comb in the slot closest to the black electrode.
   
   
3. Pour the melted agarose into gel deck.
   
   
4. Remove black casting dams and the comb.
   
   
5. Record on data sheet where you will load the DNA samples (1-4).
   
   
6. Load the DNA samples in the wells NEAR THE BLACK ELECTRODE.
   
   
7. Pour the TAE solution into the side farthest from you samples, so that you gels is completely covered plus a little more.
   
   
8. Connect electrophoresis unit to power supply. The DNA will migrate through the gel for ~30 minutes.

Methylene blue staining

1. Once the purple dye has migrated approximately 2/3 of the gel, turn off the power and carefully remove the gel.
2. The gel is very fragile, take care to not break it. You can remove the tray that you poured agarose on to and gently slide the gel into the staining tray. At this point you cannot see the DNA, what can you see and how do the four different lanes compare?
3. Once you have placed your gel into the staining tray bring it to the staining station. Completely cover the gel with methylene blue and cover try with saran wrap. Stain overnight.

Day 3 Analysis of Results:

Viewing the gel: Pour the methylene blue back into the bottle and carefully place the gel onto a white light box. The gel is very fragile so take care to not break it.

1. Diagram the DNA bands on the gel data sheet. Be as accurate as possible in drawing the bands.
   
   
2. Compare the banding pattern with a restriction enzyme map of bacteriophage lambda. Which DNA bands match with the lambda map?
   
   
3. Use semilog paper to draw a curve comparing DNA length with distance migrated. Use the Hind III digest to generate the standard curve, and determine the DNA lengths of the Eco RI DNA fragments, based on the Hind III standard curve. How do the sizes compare with the restriction enzyme map of bacteriophage lamba?
   
   

BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona August 3, 1998 Designed by: Ken Kubo, Ph.D. Last Modified March 1, 2002 Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu