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Activity 3: Restriction Enzyme digestion - How does it work? Why is it useful?

Introduction

Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms. These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of bases that they recognize. This recognition site or sequence is generally from 4 to 6 base pairs in length. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. The size of these fragments is measured in base pairs or kilobase (1000 bases) pairs.

Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested.

In the presence of specific DNA repair enzymes, DNA fragments will reanneal or stick themselves to other fragments with cut ends that are complimentary to their own end sequence. It doesn’t matter if the fragment that matches the cut end comes from the same organism or from a different one. This ability of DNA to repair itself has been utilized by scientists to introduce foreign DNA into an organism. This DNA may contain genes that allow the organism to exhibit a new function or process. This would include transferring genes that will result in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area.

In this experiment, we will use restriction enzymes to cut up DNA from a small virus called Bacteriophage λ. This virus is 48,502 base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs. Since the whole sequence of λ is already known we can predict where each restriction enzyme will cut and thus the expected size of the fragments that will be produced. If the virus DNA is exposed to the restriction enzyme for only a short time, then not every restriction site will be cut by the enzyme. This will result in fragments ranging in size from the smallest possible (all sites are cut) to in-between lengths (some of the sites are cut) to the longest (no sites are cut). This is termed a partial restriction digestion.

In this experiment, we will perform a full restriction digestion. After overnight digestion, the reaction is stopped by addition of a loading buffer. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed.

This laboratory will take approximately 3 days. The restriction digestion takes place overnight and can be kept in the freezer until the next class period when it will be be used for gel electrophoresis. The gels may be stained overnight prior to photographing or recording results.

Objectives

1. Understand what a DNA restriction enzyme is and how it works.
2. Learn to use a micropipette.
3. Learn to separate DNA on an agarose gel using electrophoresis.
4. Understand how to use a restriction digestion map to identify a sample DNA.
5. Compare the λ DNA bands on a gel to the known λ DNA restriction map.

 
Materials

For each lab group

• Four microtubes
• Microtube rack
• 20-µl micropipette (or 10-µl micropipette) and sterile tips
• Waterproof pen
• Beaker or foam cup with crushed ice for the following
  • 20 µl of 0.4 µg/µl λ DNA
  • 2.5 µl BamHI restriction enzyme
  • 2.5 µl EcoRI restriction enzyme
  • 2.5 µl HindIII restriction enzyme
• 10 µl distilled water
• Gloves
• 500-ml beaker (day 2)
• Electrophoresis chamber (day 2)
• Power supply (day 2)
• 20 µl 10X loading dye (day 2)
• 1.0% agarose gel (day 2)

Common Materials

• Container with TBE solution (1X)
• 37°C water bath w/ floating rack
• 60°C water bath or saucepan on a hot plate (day 2)
• Cooler with crushed ice
• Freezer (non frost-free, if possible)
• Camera if desired
• Distilled water
• 0.002% methylene blue stain (day 3)

Advance Preparation

Day 1:


1. If you saved the 1X TBE solution from the Gel Electrophoresis with Dyes activity, reuse it for this laboratory.
   
   
2. Obtain enough crushed ice and ice containers (styrofoam cups) for each lab group.
   
   
3. Fill a pan with water and adjust it to 55°C on a hot plate
   
   
4. Fill a second pan with water and adjust it to 37°C on a hot plate while the students complete preparation of the restriction digests.
   
   
5. Reconstitute the lambda DNA with sterile distilled water to 0.4 µg/µl.
   
   
6. Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed.
   
   
7. Make the 1.0% agarose gel solution as follows:
   
   To make 100 ml of gel, which is sufficient for 3 gels, weigh out 1.0 g of agarose and place into a 200- to 250-ml glass beaker or flask. Add 100 ml of 1X TBE (Tris-Borate-EDTA) buffer. Heat in the microwave for 30 seconds at a time, shaking gently each time, until the agarose is completely melted. Alternatively, the solution can be heated on a hot plate, with occasional gentle shaking, until the agarose is melted. Keep warm if the class will use it within a half hour. Otherwise, allow the solution to cool and solidify. Cover and keep in the refrigerator.
   
   Day 2:
   
   
8. Fill a pan with water and adjust it to 60°C.
   
   
9. Pour enough agarose gels for each lab group as follows:
   • Wear gloves
   • Microwave or warm the agarose bottle in a hot waterbath until the gel liquefies. Be sure to use a microwave designated for science purposes (not food).
   • Firmly seal the ends of the gel tray using labeling tape.
   • Place the plastic comb in the slots close to the end of the tray.
   • Pour approximately 35-40 ml of agarose into each gel tray. This will result in a thick gel so that at least 20 µl of sample can be loaded into each well.
   • Let cool until solidified (approximately 15 minutes).
   • If storing overnight, place trays in a container or ziploc baggie with 0.5X TBE solution so they do not dry out.
   
   Day 3:
   
   
10. Remove student gels from the refrigerator.
    
    
11. Set up containers for staining in a common area near a sink.

Note

Gels may be discarded in regular trash receptacle. A description of how to use a micropipet can be found in Activity 2 - Gel Electrophoresis of Dyes.

Use of Methylene Blue:

Although methylene blue dye is not as sensitive as ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment. Methylene blue is non-toxic but will stain clothes, hands, and equipment, so always wear gloves. Use the stain close to a sink and clean up spills immediately. Use distilled or deionized water to de-stain gels. Only use deionized water for making the 0.1X TBE buffer to make this stain since the high chlorine levels of most tap water will damage the DNA. A single container of methylene blue dye should be all that is needed since it may be reused several times and disposed of down the sink.

Use of Power Supplies

See description in Gel Electrophoresis of Dyes - Activity 2
Enzymes

Restriction enzymes require special care for handling and use. They lose activity unless kept frozen; exposure to warm temperatures for even a short time will result in loss of activity.

Using good sterile technique, aliquot samples for students, being careful to keep everything on ice until ready to be used.

Enzymes should be stored in a foam container in the freezer (non frost-free if available), along with the special buffer for each enzyme. The special buffers contain the salt and pH requirements for optimal activity of each enzyme.

Lambda (λ) DNA:

The λ DNA used in this laboratory can exist as either a linear or circular molecule, creating some confusion when interpreting restriction digest results. By heating the sample to 60°C for 3 minutes, immediately prior to electrophoresis, the hydrogen bonds holding the ends of the linear DNA together in a circle will be broken.

Background Reading

Since viruses have a relatively simple genome, scientists have studied their DNA and used this information to test theories and develop concepts that apply to the genetics of living organisms. One of the most studied viruses is called bacteriophage lambda (λ). Bacteriophage λ is a virus that infects bacterial cells.

Student Activity: Restriction Enzyme Analysis - Methylene Blue stain

Background Reading

Bacteriophage λ is a virus that attacks bacterial cells and is one of the most studied viruses. The information from the relatively simple virus genomes has been used to test theories and develop concepts that apply to the genetics of living organisms. The DNA of Bacteriophage λ is approximately 48,514 base pairs or 48.514 kilobase pairs in length while the human genome is approximately 3 billion base pairs.

This experiment uses special “restriction” enzymes that act as chemical scissors to cut λ DNA into pieces. Each enzyme recognizes a unique sequence of 4-6 bases along the DNA strand and cuts the strand at these sites - the first step in a process called restriction mapping. These smaller, specific sections of an organism’s DNA can then be studied in detail and an outline of the whole genome can be constructed. This procedure is one of the most important in modern biology.

The small fragments of DNA are separated by gel electrophoresis. The movement of the fragments will always be towards the positive electrode because DNA is a negatively charged molecule. The fragments move through the gel at a rate that is determined by their size and shape, with the smallest moving the fastest.

DNA cannot be seen as it moves through the gel. A loading dye must be added to each of the samples before it is pipetted into the wells. The progress of the dye can be seen in the gel. It will initially appear as a blue band, eventually resolving into two bands of different colors.

The faster moving, purplish band is bromophenol blue dye that migrates at roughly the same rate as a 300 base pair fragment of DNA. The slower moving aqua band is xylene cyanol, nearly equivalent to a 9000 base pair fragment. The faster moving band must move at least 4-7 cm from the wells to achieve the best separation of DNA for analysis. Care should be taken not to let the bromophenol blue band run off the end of the gel.

Following staining to locate the DNA, the gel is observed and the fragments appear as a pattern of bands. In this experiment, we will compare our banding pattern with a predicted result shown in figure 1.

Figure 1. Lambda DNA Restriction digest (Photo from J. Leach Laboratory)

Information may be provided by your teacher that details the process of isolating and analyzing these bands to create a DNA fingerprint.

Objectives

1. Understand what a DNA restriction enzyme is and how it works.
2. Learn to use a micropipette.
3. Learn to separate DNA on an agarose gel using electrophoresis.
4. Understand how to use a restriction digestion map to identify a sample DNA.
5. Compare the λ DNA bands on a gel to the known λ DNA restriction map.

Materials

For each lab group

• Four microtubes
• Microtube rack
• 20-µl micropipette and sterile tips
• Waterproof pen
• 250 µl distilled water
• Gloves
• 20 µl 10X loading dye (day 2)
• 1.0% agarose gel (day 2)
• Beaker or foam cups with ice for each of the following:
  • 20 µl of 0.4 µg/µl λ DNA - keep in cup of ice
  • 2.5 µl BamHI restriction enzyme - keep in cup of ice
  • 2.5 µl EcoRI restriction enzyme - keep in cup of ice
  • 2.5 µl HindIII restriction enzyme - keep in cup of ice
• 500 ml beaker (day 2)
• Colored lab tape (day 2)

• Electrophoresis chamber (day 2)
• Power supply (day 2)
• Container with TBE buffer (1X)
• 37°C water bath w/floating rack
• 60°C water bath w/floating rack
• Cooler with crushed ice
• Freezer (non-frost-free, if possible)
• Distilled water
• 0.002% methylene blue stain (day 3)
• Stain container (day 3)

Precautions

The methylene blue dye will stain skin, clothes, and equipment. Always wear gloves and safety glasses. Do all staining in a central area near the sink.

Procedure

1. Put on gloves. Keep all enzyme and DNA aliquots on ice through step 6.
   
   
2. Label 4 microtubes, reagents as indicated below, and place them in the tube rack:
   
   
   

   Reagents	BamHI	EcoRI	HindIII	Control
   10X buffer	4 µl	4 µl	4 µl	4 µl
   DNA	4.0 µl	4.0 µl	4.0 µl	4.0 µl
   BamHI	2.0 µl	0	0	0
   EcoRI	0	2.0 µl	0	0
   HindIII	0	0	2.0 µl	0
   Water	30.0 µl	30.0 µl	30.0 µl	32.0 µl

   
    

3. Set the micropipette to 4 µl and carefully add 4 µl of 10X restriction buffer to each tube. When adding the droplets of buffer to the restriction tube, touch the pipette tip to the bottom of the tube. Use a new tip for each buffer.
   
   
4. Set the micropipette to 4.0 µl and carefully add 4.0 µl of DNA to each tube, using a new tip each time.
   
   
5. Add 32.0 µl of distilled water to the control tube and 30.0 µl to the other reaction tubes.
   
   
6. Close the microtubes and heat in a 55°C waterbath for 10 minutes then immediately place on ice for 2 minutes.
   
   
7. Add 2 µl of the appropriate restriction enzyme to the reaction tubes as indicated on the grid. Use a new tip for each enzyme added.
   
   
8. Close the microtube caps and make sure that all the liquid is at the bottom of the tube by tapping the bottom of the tube gently on the desk top. Give the tubes to the instructor. They will be incubated at 37°C overnight. The tubes will then be frozen until the next class (up to 2 months).

Day 2:


1. Put on gloves. Fill a styrofoam cup with ice, collect your DNA digestion tubes and keep on ice until needed.
   
   
2. The 1.0% agarose gel will be placed into the gel box with the wells at the negative (black) end of the box.
   
   
3. Add approximately 150 ml of 1X TBE solution to the box so that the gel will be covered with about 2 mm of buffer. Remove the comb by pulling straight up, making sure that the buffer covers the gel so that it will fill the wells and help them to retain their shape as the comb is removed.
   
   
4. Heat the microtubes in a 60°C water bath for 3 minutes. This will break any hydrogen bonds holding the ends of the linear DNA together in a circle.
   
   
5. Add 4 µl of loading dye to the bottom of each of the microtubes and eject the tip into the tube. Addition of the loading dye will also stop the restriction reaction taking place in each tube. (The reaction can be stored in the refrigerator at this point for use at a later date if necessary, in this case remove the tips and close the tube caps.)
   
   
6. Set up the electrophoresis apparatus as described in Gel Electrophoresis of Dyes - Activity 2.
   
   
7. Load 20 µl of each sample into a well as shown in figure 2 above. Use the tips that were left in each tube or make sure that you use a new tip for each sample if you stored the tubes overnight. Turn on the current for about 30-45 minutes. When the purple dye from the loading dye is about 1 cm from the end of the gel, the power supply should be turned off and the gel box unplugged.
   
   
8. Place gel in a 0.002% methylene blue solution in 0.1X TBE and stain overnight at 4°C or for 2 hours at room temperature.

Day 3:


1. Observe the gel over a white light. If the bands are not visible because of a high background staining, place the gel in 0.1X TBE with gentle agitation, changing the buffer every 30-60 minutes until you are satisfied with the degree of destaining. (from http://wheat.pw.usda.gov/~lazo/methods/lazo/met1.html)
   
   
2. Photograph if desired.
   
   
3. Wash the work area thoroughly to be sure that no stain solution is left in contact with surfaces. Wash your hands!
   
   
4. Complete the activity sheet and appropriate forensics activities from either website below.

Student Activity

Restriction enzymes cut at specific sites along the DNA. These sites are determined by the sequence of bases which usually form palindromes. Palindromes are groups of letters that read the same in both the forward and backwards orientation. In the case of DNA the letters are found on both the forward and the reverse strands of the DNA. For example, the 5’ to 3’ strand may have the sequence GAATTC. The complimentary bases on the opposite strand will be CTTAAG, which is the same as reading the first strand backwards! Many enzymes recognize these types of sequences and will attach to the DNA at this site and then cut the strand between two of the bases. The restriction enzymes which we used in this laboratory are EcoRI, HindIII and BamHI and their sequences are as follows, with the cut site indicated by the arrow.

λ cut with EcoRI	λ cut with HindIII	λ cut with BamHI

This figure shows the size of each of the fragments/bands produced when λ DNA is cut with each of these restriction enzymes. The sizes were determined by comparison to a molecular ladder which has bands of known sizes when it is separated by electrophoresis at the same time as the digested λ DNA.

Restriction sites of Lambda (λ) DNA - in base pairs (bp)

The sites at which each of the 3 different enzymes will cut lambda DNA are shown in the maps Enzymes A, B and C below.

1. Calculate the size the resulting fragments will be after digestion and write them on the maps.
   
   
2. How many fragments would you expect to see for each of the maps A, B and C?
   
   
3. Draw these fragments onto the graph below.
   
   
4. Now compare the size of the fragments that you have calculated with the bands shown in the photographs of the gels and determine which of the enzymes, BamHI, EcoRI and HindIII were used to cut A, B and C.
   
   
5. How many times does the sequence GAATTC occur in the λ DNA sequence? What about AAGCTT and GGATCC?
   
   
6. Are there as many bands in your gel as you would expect to see based on the results of your calculations? If the number is different explain what you think has happened.

Answers to Student Activity

1. See map above
   
   
2. Under ideal conditions there would be 6 fragments from Enzymes A and B, and 8 fragments from Enzyme C.
   
   
3. See students graph
   
   
4. Enzyme A = BamHI
   Enzyme B = EcoRI
   Enzyme C = HindIII
   NOTE: In non-ideal conditions, the enzyme may not cut at all sites, and a partial restriction digestion will result
   
   
5. GGATCC is the recognition site for BamHI and is found in λ DNA at 5 locations.
   GAATTC is the recognition site for EcoRI and is found in λ DNA at 5 locations.
   AAGCTT is the recognition site for HindIII and is found in λ DNA at 7 locations.
   
   
6. Sometimes bands that are very close together in size will not be visible separately on these gels. There may be a single thicker band that indicates that two bands are co-localizing. When bands are very small (500 bp or less) they may have run off the end of the gel and therefore no longer be present.

Protocol adapted from http://ceprap.ucdavis.edu/Equipment/Protocols/
restriction_enzyme_analysis-methylene_blue_stain_03.pdf

Other activities:
http://www.biology.arizona.edu/human_bio/problem_sets/DNA_forensics_1/02Q.html
Use problems 2 and 3 from Problem set 1.