Materials Included In Kit

Additional Materials Required

Prelab Preparation

Electrophoresis buffer: Measure 20 mL of 50X TAE buffer in a graduated cylinder and pour into 980 mL of distilled water in a 1000-mL flask. Mix with a glass stirring rod, seal with Parafilm M or plastic wrap, and store in a refrigerator. Note: Prepare enough buffer solution to allow each group to cover the gel in the chamber to a depth of about 2 mm. Depending on the type of electrophoresis units being used, the amount of buffer needed could be as much as 300 mL per chamber. Gel preparation requires an additional 60 mL of buffer to make a 6 x 6 cm gel. Prepare fresh buffer weekly.

Methylene blue electrophoresis stain: Measure 30 mL of 10X methylene blue staining solution in a graduated cylinder and add the staining solution to 270 mL of warm distilled water in a 500-mL Erlenmeyer flask. Mix with a glass stirring rod, seal with Parafilm M or plastic wrap, and store in a refrigerator. 

Safety Precautions

Electrical Hazard: Treat these units like any other electrical source—very carefully! Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Do not try to open the lid of the unit while the power is on. Exercise caution in handling the methylene blue—it will readily stain clothing and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Remind students to wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions used in this lab may be rinsed down the drain using copious amounts of water according to Flinn Suggested Disposal Method #26b. Used gels may be placed in the regular trash according to Flinn Suggested Disposal Method #26a. The DNA in this kit is derived from bacteriophage samples. It is not pathogenic to humans and therefore is not considered a biohazard.

Lab Hints

• This laboratory activity can reasonably be completed in a 2 to 3-hr lab period. If the gels have been pre-poured and are ready to use, electrophoresis setup and sample loading will take approximately 40 minutes. The electrophoresis run itself will take 20 minutes to 2 hours depending on the voltage and equipment used. Staining and sample analysis will require an additional 45 minutes.
• Store DNA samples in the freezer until ready to use. DNA stored at room temperature or warmer may degrade over time. Short periods at room temperature will not affect results.
• Concentrated methylene blue electrophoresis stain, concentrated electrophoresis buffer, and agarose solution may be stored at room temperature.
• Gel preparation instructions are given separately for flexibility in running the lab. Gel preparation requires approximately 10–20 minutes plus at least an additional 20 minutes for the gel to solidify (60 minutes is optimum). Longer solidification times will “harden” the gel, minimizing tears and creating more distinctive bands.
• Predigested, ready-to-run DNA samples are available from Flinn Scientific. Simply pipet the DNA samples into the wells of an agarose gel and run the electrophoresis apparatus. The 80-μL sample volume per vial is enough for 6 students using 10–13 μmL per well. Ready-to-run DNA contains predigested samples, electrophoresis buffer and gel-loading solution. The gel-loading solution contains glycerol (to make the samples more dense than the buffer) and tracking dyes to follow the progress of electrophoresis separation. Two tracking dyes are used—bromphenol blue, which migrates at a rate similar to small 200–400 bp DNA fragments, and xylene cyanol, which migrates with larger, 4 kb (4000–base pair) fragments.
• When preparing agarose gels using a stirring hot plate, rotate the magnetic stir bar very slowly to diminish the number of bubbles in the agarose solution.
• Have students practice pipetting 10 μL of tap water into a practice gel while waiting their turn to load the gel. Practice gels may be prepared with less expensive agar instead of agarose.
• Run the gel at 5 V/cm. For example, if the electrodes are 25 cm apart, then the gel should be run at 125 V. Running the gel at a higher voltage may cause the agarose to melt, hindering its ability to act like a molecular sieve. Run a practice gel to determine the length of time the gel will need to run. 20–120 minutes is a reasonable range to expect results. In general, longer electrophoresis runs at lower voltages will increase the resolution of DNA fragments.
• Sample data were obtained after 42 minutes at 125 V using an Edvotek M-12 electrophoresis apparatus.
• Gel samples submerged under buffer may be stored in the refrigerator for up to two weeks.
• The recommended electrophoresis buffer is tris–acetate–EDTA (TAE). Tris–borate–EDTA (TBE) buffer may also be used. Both buffers are generally available commercially as 10X concentrates. Prepare the working strength solution by mixing one part concentrate with nine parts distilled or deionized water. Instructions for preparing the buffers from reagent chemicals may be found in your current Flinn Scientific Catalog/Reference Manual.
• Concentrated pre-cut DNA must be diluted prior to electrophoresis. The concentration of DNA fragments in commercial samples will vary by lot. Samples should be diluted with buffer according to vendor instructions so that 10 μL of sample contains at least 5 ng of each DNA fragment. (A minimum of 5 ng of DNA per band is required in order to visualize DNA using methylene blue stain.) Gel loading solution must also be added to the diluted DNA prior to loading in the wells. The amount of material loaded into a well typically includes 10 μL of diluted DNA plus 2 μL of 6X gel loading solution, for a total of 12 μL in each well. Loading too much DNA into a well will cause the bands to broaden and streak. If too little DNA is loaded into a well, the DNA bands will not be visible after staining.
• Another option for preparing DNA samples for electrophoresis is to purchase concentrated DNA, such as lambda phage DNA, and restriction enzymes separately. Concentrations of DNA restriction enzymes vary by type and lot from various biological and chemical supply houses. Flinn Scientific carries lyophilized lambda phage DNA powder as well as EcoRI and HindIII Dryzyme^® restriction enzymes. These should be stored in the freezer and reconstituted or rehydrated with sterile water and buffers prior to use. Complete instructions are provided for digestion of DNA using the restriction enzymes. The reaction temperature for the enzymes is 37 °C, and the incubation time is 60 minutes. These procedures may be done as an alternative lab prior to or in addition to this DNA forensics experiment.

Teacher Tips

• Adjustable-volume digital micropipets offer the best value for different lab activities and may be shared between the biology, chemistry, and forensic labs.
• Electrophoresis units and power supplies come in many sizes and configurations. To reduce congestion and improve flow in the teaching lab environment, we recommend the purchase of six dual electrophoresis apparatus units for a class of 24 students working in pairs. Each unit is supplied with and will accommodate two 7-cm mini-gel trays. Standard, six-well combs are also included.

Answers to Prelab Questions

1. List the safety precautions that must be followed when performing electrophoresis.

   Ensure that the work surface, connecting wires and terminals are all dry. Do not open the electrophoresis chamber while the power is on. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron.

2. Explain the function of each of the following components of gel electrophoresis:
   1. Agarose gel—The agarose gel is the microscopic “strainer” that separates big molecules from small ones because of the size of the pores within the agarose.
   2. Electrophoresis buffer—This acts as a liquid conductor that carries the electric current. It also maintains a stable pH.
   3. Wells in the gel—These hold the samples that will travel through the gel.
   4. Electric current—Provides the force that moves charged molecules (DNA fragments) through the gel.
3. Briefly summarize how gel electrophoresis is used to separate molecules.

   Electrophoresis involves the use of an agar-like material that acts as molecular filter paper. When samples are placed in wells within the gel and the electricity is turned on, the resulting electric field causes charged molecules, which make up biological samples, to move through the gel and separate according to their charge, size and shape.

4. Lambda phage DNA is 48,502 base-pairs (bp) long and has five recognition sites for the EcoRI restriction enzyme. Starting from the 5'-end, the sites occur at 21,226 bp; 26,104 bp; 31,747 bp; 39,168 bp; and 44,972 bp. How many fragments will be obtained when lambda phage DNA is digested with EcoRI? List the fragment size for each. Hint: To visualize the fragmentation process, draw a line to represent a DNA strand and then mark off the restriction sites.

   Six fragments a–f should be obtained, with fragment lengths from largest to smallest corresponding to 21,226 bp; 7,421 bp; 5,804 bp; 5,643 bp; 4,878 bp; and 3,530 bp.

   {14046_PreLabAnswers_Figure_4}

   a = 21,226 bp
   b = 26,104 – 21,226 = 4,878 bp
   c = 31,747 – 26,104 = 5,643 bp
   d = 39,168 – 31,747 = 7,421 bp
   e = 44,972 – 39,168 = 5,804 bp
   f = 48,502 – 44,972 = 3,530 bp

Answers to Questions

1. Sketch the approximate locations of the DNA bands in the following graphic. Sample DNA Banding Pattern
   {14046_Answers_Figure_5}
2. Using a metric ruler, measure the migration distance in millimeters for each major band and enter the results in the following table.
   {14046_Answers_Table_1}

   *The DNA ladder will contain 10–14 visible bands. List the migration distances separately for the additional bands in the DNA ladder. For the DNA 1-kb ladder, additional bands should appear at 75, 81, 86, 92, 97 and 103 mm. There may also be four closely spaced fragments at 108–116 mm.

   Migration distances will vary due to differences in the electrophoresis run and periodic substitution of DNA. All DNA fragments should be between 75 and 24,000 bp.

3. Evaluate the banding patterns in the electrophoresis experiment and identify any matching DNA samples.

   DNA Samples 3 and 4 match.

4. List three errors that could affect the outcome of any gel electrophoresis procedure.
   1. Not placing the sample deep enough into the well or not placing enough sample into the well. .
   2. Puncturing the well with the pipet tip causing the dye to actually run below the gel. .
   3. Connecting the wires to the power supply or chamber incorrectly. .
   4. Contaminating pure samples by using the same pipet tip in different samples. .
   5. Not recording which sample went into which well.
5. Why would a forensic scientist use the polymerase chain reaction technique to prepare DNA samples for analysis?

   PCR is used when forensic scientists need to amplify the amount of DNA obtained in very small amounts from a crime scene.

6. The following table lists the migration distance and fragment size for Lambda DNA cut by the restriction enzyme HindIII and analyzed on a 0.8% agarose gel at 70 V. Plot the data on the semi-log graph shown below. Draw a smooth curve through the points and explain how the graph could be used to determine the fragment size for an unknown band. Estimate the base-pair fragment size for a band that appears at 30 mm in the same gel.
   {14046_Answers_Table_2}

   A graph of the log of the fragment size versus migration distance gives a smooth curve that can be used as a calibration curve to determine the size of an unknown fragment if its migration distance is measured on the same gel. (Conditions must be identical for this comparison to be valid.) A fragment appearing at 30 mm would be approximately 3,600 bp in length.

   {14046_Answers_Figure_6}

Teacher Handouts

14046_Teacher1.pdf

Introduction

The world of forensic science was revolutionized with the discovery of scientific techniques for identifying humans—and, indeed, all living things—using DNA. DNA fingerprinting can be used to identify the source of DNA in forensic investigations and has also been used to diagnose genetic diseases, identify disaster victims and study evolutionary relationships among organisms.

Concepts

• Gel electrophoresis
• Restriction fragment length polymorphism (RFLP)
• Genes
• Variable number of tandem repeats (VNTR)
• Polymerase chain reaction (PCR)
• Restriction enzymes

Background

Gel electrophoresis is an analytical method for the separation, identification and analysis of biological molecules, including DNA, RNA and proteins, in an electric field. In 1955, the British-born American scientist Oliver Smithies determined that when a colloidal gel made of starch was positioned between positive and negative electrodes, it acted like a molecular “sieve” for the macromolecules. Proteins with different sizes, shapes and molecular charge moved through the gel at different rates, with smaller molecules or fragments moving faster through the maze of microscopic pores. Charged proteins always moved toward the electrode with the opposite charge. For example, a negatively charged protein migrates through a gel toward the positive electrode, which is called the anode. DNA sequencing methods have been built upon the original electrophoresis principles developed by Smithies, who received the Nobel Prize for medicine in 2007 for innovations, including specific gene modifications in mice, that revolutionized genetic research.

In 1984, Sir Alec Jeffreys of the University of Leicester in England published the first account of the basic principles, techniques and applications of DNA fingerprinting. Combining his interest in human genetics with research experience in molecular biology probing mammalian genes, Sir Jeffreys had been studying the production and separation of DNA fragments to detect variations between individuals and identify the causes of inherited diseases. He had earlier found that when DNA was hydrolyzed or cut into fragments using so-called restriction enzymes and separated using electrophoresis, the results yielded distinctive banding patterns due to the formation of different-size DNA fragments. This result, called restriction fragment length polymorphism, or RFLP, arose due to variations in DNA sequences between individuals. Although RFLP showed differences in the pattern of enzyme recognition sites, it produced far too many bands and was therefore too crude or blunt a technique to be useful for the unique identification of individuals. By focusing on one gene of interest, and hybridizing part of it to DNA fragments lifted from electrophoresis analysis of DNA of different individuals, Jeffreys found more subtle variations in the number of repeating DNA base-pair sequences between genes on a chromosome. Every living thing, except identical twins, has a unique number of tandem DNA repeats called variable number of tandem repeats (VNTR). Since these tandem repeats are not part of a gene, they do not affect the viability of an organism. The basic procedures involved in DNA fingerprinting are described.

In order to analyze DNA, it must first be extracted from the cells of an organism—any cell that contains a nucleus can be used to identify DNA. The sample of cells is homogenized in a blender or crushed in a mortar with salt and a detergent to break the cell membranes and expose the DNA. An enzyme is added to the mixture to facilitate the uncoupling of the DNA from its histones (DNA proteins). When alcohol is added on top of the treated cell mixture, the DNA becomes insoluble and moves to the alcohol layer, where it can be easily retrieved.

At this point, the DNA molecules are too long to be analyzed by electrophoresis. The DNA must first be fragmented at very specific base-pair locations using one or more restriction enzymes. Enzymes that break DNA molecules at internal positions are called restriction endonucleases. Enzymes that degrade DNA by digesting the molecule from the ends of the DNA strand are termed exonucleases. More than 2500 different restriction enzymes are available to molecular biologists to analyze DNA. Each restriction enzyme recognizes a specific nucleotide sequence. The enzyme “scans” the length of a DNA molecule and then digests it (breaks it apart) at or near a particular recognition sequence. The specific sequence may be five to sixteen basepairs long. For example, the EcoRI endonuclease has the following six–base-pair recognition sequence:

EcoRI breaks the double-stranded DNA at the locations indicated by the dotted line and produces “ragged-ended” sequences, often called sticky ends. Other endonucleases cut the DNA cleanly at one specific base-pair, producing what are called blunt ends. Restriction endonucleases are named using the following convention:

EcoRI

E = genus Escherichia
co = species coli
R = strain RY 13
I = first restriction enzyme to be isolated from this species

The first letter (capitalized) indicates the genus of the organism from which the enzyme was isolated. The lower case letters that follow indicate the species. Additional letters indicate the particular strain used to produce the enzyme. The Roman numeral denotes the sequence in which enzymes from a particular species and strain of bacteria were isolated.

Since the average human DNA sequence is more than 3.2 billion base-pairs long (about 20,000 genes), there may be as many as 750,000 fragments of DNA after a single restriction enzyme completes the fragmentation of a single cell’s DNA. RFLP fragments created by the use of one or more restriction enzymes are loaded into an agarose gel in an electrophoresis chamber. Agarose is a refined form of agar that is made from seaweed. The agarose gel is positioned between two electrodes with the wells located by the cathode (negative electrode). This allows the negatively charged DNA to move toward the anode (positive electrode). The electrophoresis chamber is filled with a buffer solution, bathing the gel in a solution that shields the system from changes in pH.

DNA fragments are white to colorless and appear invisible in the gel. Colored tracking dyes are added to the DNA sample to monitor the progress of the sample as it moves through the gel. Typically, two dyes are added, one that migrates at a rate similar to the smaller DNA fragments and another that migrates at a rate similar to the largest DNA fragments. Once the first dye migrates to within 1 cm of the end of the gel, the power is shut off to the electrophoresis unit. The DNA stops migrating since the electromotive force stops.

The agarose gel is then removed from the electrophoresis chamber and transferred to a staining tray. The stain binds to the DNA fragments revealing a banding pattern. Molecular biologists use a radioactive stain and X-ray film to visualize the banding pattern. In this experiment a colored dye that may be viewed with visible light will be used. The banding pattern is unique since the DNA sample is unique to each individual or organism, except identical twins or asexual offspring of less complex organisms. The banding pattern is measured against a series of known DNA standards and samples prepared and analyzed with the unknown DNA sample. For example, paternity test runs include a known standard of human DNA plus samples of the mother, likely fathers, and that of the child. A match is determined by calculating the probability of an individual having a particular combination of bands in a population. The more matching bands that are observed, the more likely it is that the DNA comes from the same person.

A second sample may be run using a different restriction enzyme. A different banding pattern will be revealed for the same DNA samples because the DNA sequence is cleaved at different base-pair locations. With the exception of identical twins who share identical genotypes, the probability of two individuals having identical banding patterns for two series of DNA cuts is less than the current human population. The theoretical risk of a coincidental match has been estimated at 1 in 100 billion.

The mobility of negatively charged DNA fragments in an electrophoresis experiment may be standardized by running the fragments repeatedly under identical conditions (i.e., pH, voltage, time, gel type, gel concentration). Under identical conditions, identical-length DNA fragments will move the same distance in a gel. Thus, the size of an unknown DNA fragment can be determined by comparing distance on an agarose gel with that of DNA marker samples of known size. The size of the DNA fragment is usually given in nucleotide base-pairs (bp). The smaller the DNA fragment, the faster it will move through the gel during electrophoresis.

Good scientific protocol is critical to the outcome of any laboratory work, especially in forensic analysis. Sloppy work might convict the wrong person or let a guilty suspect go free. Consequently, analysts must carefully document which restriction enzyme was used, the conditions and chemicals that were used, and the names of all known standards and controls that were prepared with the DNA sample.

RFLP is limited by the quantity of DNA available and the degree of degradation of the sample. Modern techniques in DNA forensic analysis utilize the polymerase chain reaction (PCR). This technology can be used to amplify (make numerous exact duplicates of) as little as a single molecule of DNA. The amplified sample may then be digested by restriction enzymes, electrophoresed, stained, and analyzed. PCR technology is able to analyze any detectable DNA sample, no matter how small or degraded the original sample.

Experiment Overview

The purpose of this activity is to demonstrate the basic principles of DNA forensics using gel electrophoresis. The process will be used to identify matching DNA profiles from a collection of DNA samples.

Materials

Prelab Questions

1. List the safety precautions that must be followed when performing electrophoresis.
2. Explain the function of each of the following components of gel electrophoresis:
   1. Agarose gel
   2. Electrophoresis buffer
   3. Wells in the gel
   4. Electric current
3. Briefly summarize how gel electrophoresis is used to separate molecules.
4. Lambda phage DNA is 48,502 base-pairs (bp) long and has five recognition sites for the EcoRI restriction enzyme. Starting from the 5'-end, the sites occur at 21,226 bp; 26,104 bp; 31,747 bp; 39,168 bp; and 44,972 bp. How many fragments will be obtained when lambda phage DNA is digested with EcoRI? List the fragment size for each. Hint: To visualize the fragmentation process, draw a line to represent a DNA strand and then mark off the restriction sites.

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Use heat protective gloves and eye protection when handling hot liquids. Methylene blue will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Loading a Gel

1. Assemble the electrophoresis unit according to the manufacturer’s instructions.
2. Make sure the electrophoresis chamber is on a level surface and do not move the unit after loading the samples.
3. Gently slide a prepared gel from a resealable bag into the casting tray with the wells toward the cathode (–) end of the unit.
4. Carefully position the gel and tray in the electrophoresis chamber. Caution: Be careful not to break or crack the gel. If the gel is damaged, it should not be used as cracks will affect the results.
5. Pour enough electrophoresis buffer into the unit to submerge the entire gel surface to a depth of 2–5 mm.
6. By convention, DNA gels are read from left to right, with the wells located at the top of the gel. With the gel lined up in the chamber and the wells located to the left, load the contents of DNA Sample 1 into the well closest to you. When the gel is turned so that the wells are at the top, “1” will be in the upper left corner.
7. Shake the microcentrifuge tubes containing DNA samples and lightly tap the bottom of each tube to mix the contents.
8. Withdraw 10 μL of DNA Sample 1 from the microcentrifuge tube using a digital micropipet.
9. Dispense the sample into the first well by holding the pipet tip just inside the well. The sample will sink to the bottom of the well. Caution: Do not puncture the bottom or sides of the well. Do not draw liquid back into the pipet after dispensing the sample (see Figure 1).
   {14046_Procedure_Figure_1}
10. Using a fresh pipet tip, withdraw 10 μL of DNA Sample 2 and load it into well 2, adjacent to DNA Sample 1.
11. Repeat step 10 for the remaining DNA samples. Use a clean pipet tip for each sample. The DNA reference ladder should go into well 5.

Running the Gel

1. Place the lid on the electrophoresis chamber and connect the unit to the power supply.
2. Run the gel as directed by the instructor. Bubbles will be visible along the electrodes while the sample is running. The bubbles are due to the electrolytic decomposition of water—hydrogen at the cathode and oxygen at the anode.
3. Turn off the power when the first tracking dye is 1 cm from the positive end of the gel. (This may take 30 minutes to 2 hours.) The time necessary to run a gel depends on the apparatus and the applied voltage.
4. When the power is off, remove the cover and carefully remove the gel tray from the chamber. Place the gel tray on a paper towel, being careful not to break or crack the gel.

Staining and Analyzing the Gel
For best results, stain the gel immediately and then destain.

1. Slide the gel off the tray and into the staining tray. Note: Do not stain the gel tray.
2. Gently pour 40 mL of the methylene blue staining solution into the staining tray. Allow the gel to stain for at least 5–10 minutes.
3. Pour off the stain into a glass beaker. The stain may be reused by other groups.
4. To destain the gel, gently pour room temperature distilled water into the staining container. Note: Do not exceed 37 °C; warmer water may soften the gel. Occasionally agitate the water and destain for 10 minutes.
5. Pour off the water into a waste beaker and repeat step 4 until DNA bands are visible.
6. Record the relative locations of each band in the Laboratory Report. The gel may be placed on a light box to visualize faint bands.

Student Worksheet PDF

14046_Student1.pdf