Activity 3: Analysis of restriction enzymes (2023)

Activity 3: Restriction enzyme digestion: how does it work? Why is it useful?

Introduction

special enzymes calledrestrictive enzymethey have been discovered in many different bacteria and other single-celled organisms. These restriction enzymes can search a length of DNA for a specific sequence of bases that they recognize. This recognition site or sequence is generally 4 to 6 base pairs in length. Once located, the enzyme binds to the DNA molecule and cuts each strand of the double helix. The restriction enzyme will continue to do this throughout the DNA molecule, which will then be broken into fragments. The size of these fragments is measured in base pairs or kilobase pairs (1000 bases).

Since the recognition site or base pair sequence for each restriction enzyme is known, we can use it to create a detailed base sequence analysis in specific regions of DNA of interest.

In the presence of certainDNA-reparaturaenzima, the DNA fragments are reassembled or ligated to other fragments with cut ends complementary to their own final sequence. It does not matter if the fragment corresponding to the cut end comes from the same organism or from another. Scientists have used this ability of DNA to repair itself to introduce foreign DNA into an organism. That DNA may contain genes that allow the organism to exhibit a new function or process. This includes the transfer of genes that result in a change in the nutritional quality of a crop or allow a plant to grow in a colder region than its usual preferred area.

In this experiment we use restriction enzymes to cut the DNA of a small virus called a bacteriophage λ. This virus is 48,502 base pairs long, which is very small compared to the human genome, which is about 3 billion base pairs long. Since the full sequence of λ is already known, we can predict where each restriction enzyme will cut, and thus the expected size of the fragments produced. If the viral DNA is only briefly exposed to the restriction enzyme, the enzyme will not cut all the restriction sites. This results in chunks ranging in size from the smallest possible (all locations are cut off) to intermediate lengths (some of the locations are cut off) to the longest (no locations are cut off). This is denoted as apartial restriction digestion.

In this experiment, we perform a complete summary of constraints. After overnight digestion, the reaction is stopped by adding loading buffer. The DNA fragments are separated by electrophoresis, a process in which an electric field is applied to cause the DNA fragments to migrate on an agarose gel. The gel is then stained with methylene blue to reveal the DNA bands and can be photographed.

This lab lasts about 3 days. Restriction digestion is carried out overnight and if used for gel electrophoresis it can be stored in the freezer until the next class. Gels can be stained before photographing or results recorded overnight.

Goals

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

materials

For each laboratory group

• four microtubes
• microtube holder
• 20 µL micropipette (or 10 µL micropipette) and sterile tips
• waterproof pen
• Glasses or foam cups filled with crushed ice for the following
  • 20 µl of 0.4 µg/µl of λ-DNA
  • 2.5ulBamHI restriction enzyme
  • 2.5ulecologicalRI restriction enzyme
  • 2.5ulRearrestriction enzyme III
• 10µl distilled water
• gloves
• 500 ml glass (day 2)
• Electrophoresis chamber (day 2)
• Power supply (day 2)
• 20 µl of 10X loading corant (day 2)
• 1.0% agarose gel (day 2)

Common Materials

• Fill with TBE solution (1X)
• Bain-marie at 37°C with floating rack
• Bain-marie at 60°C or casserole on fire (day 2)
• cold box with crushed ice
• Freezer (preferably not frost free)
• camera if you want
• Distilled water
• Staining with 0.002% methylene blue (day 3)

preparation in advance

Label 1:
1. If you saved the 1X TBE solution from the stain gel electrophoresis activity, reuse it in this lab.
2. Obtain crushed ice and enough ice cubes (styrofoam cups) for each lab group.
3. Fill a pot with water and heat it to 55°C on a hot plate.
4. Fill a second pot with water and place at 37°C on a hot plate while students complete the preparation of the constraint summaries.
5. Reconstitute lambda DNA at 0.4 µg/µl with sterile distilled water.
6. Aliquot lambda DNA, enzymes, and loading dye for each pool and store in the freezer until use.
7. Prepare the 1.0% agarose gel solution as follows:

   To prepare 100 mL of gel, enough for 3 gels, weigh out 1.0 g of agarose and place in a 200-250 mL beaker or bottle. Add 100 mL of 1X TBE (Tris-Borate-EDTA) buffer. Microwave for 30 sec at a time, gently swirling each time, until the agarose is completely melted. Alternatively, the solution can be heated on a hot plate with occasional gentle agitation until the agarose is melted. Stay warm when the gang uses it in half an hour. If not, allow the solution to cool and solidify. Cover and store in the refrigerator.

   Label 2:

8. Fill a saucepan with water and adjust to 60°C.
9. Pour enough agarose gels into each lab pool as follows:
   • use luvas
   • Heat the vial of agarose in the microwave or in a water bath until the gel liquefies. Be sure to use a microwave designed for scientific (non-food) use.
   • Seal the ends of the gel tray tightly with masking tape.
   • Insert the plastic comb into the slots near the end of the tray.
   • Pour approximately 35-40 ml of agarose into each gel tray. This creates a thick gel that allows at least 20 µl of sample to be loaded into each well.
   • Let cool until firm (about 15 minutes).
   • If storing overnight, place the peels in a container or ziploc bag filled with 0.5X TBE solution to prevent them from drying out.
   
   Label 3:
   
   
10. Take the student gels out of the refrigerator.
11. Place paint containers in a common area near a sink.

To use

The gels can be disposed of in the normal waste container. For a description of how to use a micropipette, seeactivity 2- Stain gel electrophoresis.

Methylene Blue Uses:

Although methylene blue staining is not as sensitive as ethidium bromide, it can be used to stain the larger amounts of DNA used in this experiment. Methylene blue is non-toxic, but it will stain clothing, hands, and equipment, so always wear gloves. Use the stain near a sink and clean up spills immediately. Use distilled or deionized water to decolorize the gels. Use only deionized water to prepare TBE 0.1X buffer to prepare this stain, as the high chlorine content of most tap water damages DNA. A single container of methylene blue food coloring should be all it takes, as it can be reused multiple times and flushed down the drain.

Use of power supplies.

See description in Stain Gel Electrophoresis -activity 2
Difficult

Restriction enzymes require special care in their handling and use. They lose activity unless they are frozen; are exposed to high temperatures, even for a short period of timeloss of activity.

Using good sterile technique, aliquot samples to students, making sure to keep everything on ice until ready to use.

Enzymes should be stored in a foam container in the freezer (not frost free, if available) along with the specific buffer for each enzyme. Special buffers contain the salt and pH requirements for optimal activity of each enzyme.

Lambda (λ)-DNA:

The λ-DNA used in this lab can exist as a linear or circular molecule, which causes some confusion when interpreting restriction digest results. Heat the sample at 60 °C for 3 minutes immediately before electrophoresis breaks the hydrogen bonds that hold the ends of linear DNA together in a circular pattern.

read deep

Because viruses have relatively simple genomes, scientists have studied their DNA and used this information to test theories and develop concepts applicable to the genetics of living organisms. One of the best-studied viruses is the lambda (λ) bacteriophage. The bacteriophage λ is a virus that infects bacterial cells.

Student Activity: Restriction Enzyme Analysis: Methylene Blue Staining

read deep

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

This experiment uses special "restriction" enzymes that act like chemical scissors to cut λ-DNA into pieces. Each enzyme recognizes a unique sequence 4 to 6 bases along the DNA strand and cuts the strand at those points, the first step in a process called restriction mapping. These smaller specific sections of an organism's DNA can be examined in detail, creating a blueprint of the entire genome. This procedure is one of the most important in modern biology.

Small DNA fragments are separated by gel electrophoresis. The fragments will always move towards the positive electrode, since DNA is a negatively charged molecule. The fragments move through the gel at a speed 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 sample before pipetting into the wells. The staining course can be seen in the gel. Initially it appears as a blue band, which eventually resolves into two bands of different colors.

The fastest moving purple band is bromophenol blue dye, which travels at about the same speed as a 300 base pair fragment of DNA. The slowest moving aquatic band is xylene cyanol, roughly equivalent to a 9,000 base pair fragment. The fastest moving band should be at least 4-7 cm from the wells to obtain the best DNA separation for analysis. Care must be taken that the bromophenol blue band does not run to the end of the gel.

After staining to locate the DNA, the gel is viewed and the fragments appear as a pattern of bands. In this experiment, we will compare our fringe pattern to a predicted result shown in Figure 1.

Figure 1. Restriction digest of lambda DNA (photo by J. Leach Laboratory)

Your teacher can provide information describing the process of isolating and analyzing these bands to create a DNA fingerprint.

Goals

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

materials

For each laboratory group

• four microtubes
• microtube holder
• 20 µL micropipette and sterile tips
• waterproof pen
• 250µl distilled water
• gloves
• 20 µl of 10X loading corant (day 2)
• 1.0% agarose gel (day 2)
• Cups or ice foam cups for each of the following:
  • 20 µl 0.4 µg/µl λ DNA - store in ice cream cup
  • 2.5ulBamRestriction enzyme HI - store in sundae
  • 2.5ulecologicalRI restriction enzyme - store in sundae
  • 2.5ulRearIII Restriction enzyme - store in sundae
• 500 ml cup (day 2)
• Color Lab Tape (Day 2)

Common Materials

• Electrophoresis chamber (day 2)
• Power supply (day 2)
• Contenedor con TBE-Puffer (1X)
• Bain-marie at 37°C with floating rack
• Bain-marie at 60°C with floating rack
• cold box with crushed ice
• Freezer (not frost free if possible)
• Distilled water
• Staining with 0.002% methylene blue (day 3)
• Tincture bottle (day 3)

Precautions

Methylene blue dye stains skin, clothing, and equipment.🇧🇷 Always wear gloves and safety glasses. Do all the coloring in a central area near the sink.

process

1. Put on the gloves.Keep all enzyme and DNA aliquots on ice until step 6.
2. Label 4 microtubes with reagents as follows and place them in the tube rack:

   reagents	BamHOLA	ecologicalRhode Island	Rearthird	to control
   10 blowers.	4 ul	4 ul	4 ul	4 ul
   DNS	4,0ul	4,0ul	4,0ul	4,0ul
   BamHOLA	2,0ul	0	0	0
   ecologicalRhode Island	0	2,0ul	0	0
   Rearthird	0	0	2,0ul	0
   agua	30,0ul	30,0ul	30,0ul	32,0ul

3. Set the micropipette to 4 µL and carefully add 4 µL of 10X Restriction Buffer to each tube. When adding drops of buffer to the restriction tube, gently tap the bottom of the tube with the tip of the pipettor. Use a new tip for each tampon.
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. Cap the microtubes and heat in a 55°C water bath for 10 min, then immediately place on ice for 2 min.
7. Add 2 µl of the appropriate restriction enzyme to the reaction tubes as indicated by the grid. Use a new tip for each enzyme added.
8. Close the lids of the microtubes and ensure that all the liquid is in the bottom of the tube by gently tapping the bottom of the tube on the table. Give the tubes to the teacher. They are incubated overnight at 37°C. The tubes are then frozen until the next class (up to 2 months).

Label 2:
1. Put on the gloves. Fill a Styrofoam cup with ice, collect your DNA digestion tubes, and keep them on ice until needed.
2. The 1.0% agarose gel is placed in 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, covering the gel with approximately 2 mm of buffer. Remove the comb by pulling it up, making sure the plug covers the gel to fill the wells and help them retain their shape when you remove the comb.
4. Heat the microtubes in a water bath at 60°C for 3 minutes. This breaks all the hydrogen bonds that hold the ends of linear DNA together in a circle.
5. Add 4 µL of Loading Dye to the bottom of each microtube and place the tip in the tube. The addition of the loading dye also stops the restriction reaction occurring in each tube. (The reaction can be refrigerated at this point for possible later use. In this case, remove the tips and close the lids of the tubes.)
6. Set up the electrophoresis device as described in Stain Gel Electrophoresis -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 be sure to use a new tip for each sample if you stored the tubes overnight. Turn on the power for about 30-45 minutes. When the purple dye loading dye is about 1 cm from the end of the gel, the power should be turned off and the gel box unplugged.
8. Spot gel in 0.002% methylene blue solution in 0.1X TBE and stain overnight at 4°C or 2 hours at room temperature.

Label 3:
1. Observe the gel under a white light. If the bands are not visible due to strong background staining, place the gel in 0.1X TBE with gentle agitation and change the buffer every 30 to 60 min until you are satisfied with the level of discoloration. (Outsidehttp://trigo.pw.usda.gov/~lazo/methods/lazo/met1.html)
2. Photography if you wish.
3. Thoroughly wash the work area to ensure that no staining solution is left in contact with the surfaces. Wash your hands
4. Complete the activity sheet and the corresponding forensic activities at one of the following websites.

student activity

Restriction enzymes cut at specific points along the DNA. These sites are determined by the sequence of bases that are normally formedpalindrome🇧🇷 Palindromes are groups of letters that sound the same both forwards and backwards. In the case of DNA, the letters are on the forward and reverse DNA strands. For example, the 5' to 3' strand may have the sequence GAATTC. The complementary bases on the opposite strand are CTTAAG, which is the same as reading the first strand backwards. Many enzymes recognize this type of sequence and attach to the DNA at that point and then cut the strand between two of the bases. The restriction enzymes that we use in this laboratory areecologicalRHODE ISLAND,RearIII miBamHI and its sequences are as follows, with the cleavage site indicated by the arrow.

λ cut withecologicalRhode Island	λ cut withRearthird	λ cut withBamHOLA

This figure shows the size of each of the fragments/bands generated when λ-DNA is cut with each of these restriction enzymes. Sizes were determined by comparison with a molecular ladder that exhibits bands of known size when electrophoresed simultaneously with digested λ-DNA.

Lambda DNA restriction sites (λ) - in base pairs (bp)

The locations where each of the 3 different enzymes cut lambda DNA are shown in enzyme maps A, B, and C below.

1. Calculate the size of the resulting fragments after digestion and plot them on maps.
2. How many fragments would you expect for each of maps A, B, and C?
3. Plot these fragments in the box below.
4. Now compare the fragment sizes you calculated with the bands shown in the images of the gels and determine which of the enzymes,BamHOLA,ecologicalRI eRearIII were used to cut A, B and C.
5. How often does the GAATTC sequence occur in the λ DNA sequence? What about AAGCTT and GGATCC?
6. Are there as many bands in your gel as you would expect based on the results of your calculations? If the number is different, explain what you think happened.

Answers to student activities.

1. see map above
2. Under ideal conditions, there would be 6 fragments of enzymes A and B and 8 fragments of enzyme C.
3. See student table
4. Enzyme A =BamHOLA
   Enzyme B =ecologicalRhode Island
   Enzyme C =Rearthird
   NOTE: Under non-ideal conditions, the enzyme may not be cut in all places and a partial restriction digest may occur.
5. GGATCC is the detection authority forBamHI and is located at 5 sites in λ-DNA.
   GAATTC is the recognition site forecologicalRI and is located at 5 sites in λ-DNA.
   AAGCTT is the detection site forRearIII and is found at 7 sites in λ-DNA.
6. Sometimes very close bands are not separately visible in these gels. There may be a single thicker band indicating that two bands are located in the same place. If the bands are very small (500 bp or less), they may have oozed towards the end of the gel and are therefore no longer present.