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.

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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

Understand what a DNA restriction enzyme is and how it works. Learn to use a micropipette. Learn to separate DNA on an agarose gel using electrophoresis. Understand how to use a restriction digestion map to identify a sample DNA. 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 enzyme10 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: If you saved the 1X TBE solution from the Gel Electrophoresis with Dyes activity, reuse it for this laboratory. Obtain enough crushed ice and ice containers (styrofoam cups) for each lab group. Fill a pan with water and adjust it to 55°C on a hot plate 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. Reconstitute the lambda DNA with sterile distilled water to 0.4 µg/µl. Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed. 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: Fill a pan with water and adjust it to 60°C. 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:Remove student gels from the refrigerator. 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 2Enzymes

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

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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.

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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.

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Objectives

Understand what a DNA restriction enzyme is and how it works. Learn to use a micropipette. Learn to separate DNA on an agarose gel using electrophoresis. Understand how to use a restriction digestion map to identify a sample DNA. 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 ice500 ml beaker (day 2) Colored lab tape (day 2) Common Materials 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

Put on gloves. Keep all enzyme and DNA aliquots on ice through step 6. Label 4 microtubes, reagents as indicated below, and place them in the tube rack:
ReagentsBamHIEcoRIHindIIIControl
10X buffer4 µl4 µl4 µl4 µl
DNA4.0 µl4.0 µl4.0 µl4.0 µl
BamHI2.0 µl000
EcoRI02.0 µl00
HindIII002.0 µl0
Water30.0 µl30.0 µl30.0 µl32.0 µl

 

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. Set the micropipette to 4.0 µl and carefully add 4.0 µl of DNA to each tube, using a new tip each time. Add 32.0 µl of distilled water to the control tube and 30.0 µl to the other reaction tubes. Close the microtubes and heat in a 55°C waterbath for 10 minutes then immediately place on ice for 2 minutes. 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. 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).