Electrophoresis Lab 2023

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Oct 30, 2023

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Introduction to Gel Electrophoresis “ DNA Profiling” I ntroduction Technicians working in forensic labs are often asked to do DNA profiling or “fingerprinting” to analyze evidence in law enforcement cases and other applications. Restriction Fragment Length Polymorphism (RFLP) has been the workhorse of forensic DNA profiling for many years. First described by English geneticist Alec Jeffries in 1985, RFLP analysis provides a unique banding pattern based on the restriction sites present in an individual’s DNA sequence. Currently, DNA profiling involves polymerase chain reaction (PCR) amplifi- cation which allows the analysis of minute quantities of DNA in a much shorter time. In this lab activity, you will compare band patterns produced by restriction enzyme cleavage of DNA samples when separated on an agarose gel (RFLP). The patterns in this exercise are produced from one sample that represents DNA taken at the crime scene and five samples obtained from suspects in the case. As a person’s Restriction Fragment Length Polymorphism is inherited as a unique combination of sequences from his or her parents, theoretically no two individuals will produce the same RFLP pattern in gel electrophoresis analysis. This laboratory exercise models the more elaborate technique that is performed on complex human DNA samples. Human DNA is not used in this simulation. Restriction Enzymes Scientists have benefited from a natural, bacterial defense mechanism: the restriction enzyme . A restriction enzyme acts like molecular scissors, making cuts at specific sequence of base pairs that it recognizes. These enzymes destroy DNA from invading viruses, or bacteriophages (phages). Phages are viruses that infect and destroy bacteria. Bacterial restriction enzymes recognize very specific DNA sequences within the phage DNA and then cut the DNA at that site. Although the bacteria’s own DNA may also contain these sites the bacteria protect their own restriction sites by adding a methyl group. Once purified in the laboratory the fragmented phage DNA no longer poses a threat to the bacteria. These restriction endonucleases (endo = within, nuclease = enzyme that cuts nucleic acids) are named for the bacteria from which they were isolated. For example, Eco RI was isolated from Escherichia coli . Restriction enzymes can be used to cut DNA isolated from any source. Restriction enzymes were named before scientists understood how they functioned because they would limit (or restrict) the growth of phages. A restriction enzyme sits on a DNA molecule and slides along the helix until it recognizes specific sequences of base pairs that signal the enzyme to stop sliding. The enzyme then cuts or chemically separates the DNA molecule at that site—called a restriction site . If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites, resulting in multiple fragments. Therefore, if a linear piece of DNA is cut with a restriction enzyme whose specific recognition site is found at two different locations on the DNA molecule, the result will be three fragments of different lengths. The length of each fragment will depend upon the location of restriction sites on the DNA molecule. DNA that has been cut with restriction enzymes can be separated and observed using a process known as agarose gel electrophoresis . The term electrophoresis means to carry with electricity. Lab instructions adapted from Biotechnology Explorer™, Forensic DNA Fingerprinting Kit Instruction Manual, Catalog #1666-007EDU, Bio-Rad Life Science, 2000 Alfred Nobel Drive, Hercules, CA 94547. Duplication is permitted for classroom use only.

Agarose Gel Electrophoresis Agarose gel electrophoresis separates DNA fragments by size. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are nega- tively charged, they will be drawn toward the positive pole (anode) when placed in an electric field (see figure 1 next page). The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is inversely proportional to its size in base pairs. Over a period of time, smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in single bands of DNA. These bands will be seen in the gel after the DNA is stained. Consider this analogy. Imagine that all the desks and chairs in the classroom have been randomly pushed together. An individual student can wind his/her way through the maze quickly and with little difficulty, whereas a string of four students would require more time and have difficulty working their way through the maze. DNA Fingerprinting Each person has similarities and differences in DNA sequences. To show that a piece of DNA contains a specific nucleotide sequence, a radioactive complementary DNA probe can be made that will recognize and bind that sequence. Radioactive probes allow molecular biologists to locate, identify, and compare the DNA of different individuals. This probe can be described as a “radioactive tag” that will bind to a single stranded DNA fragment and produce a band in a gel or a band on a piece of blotting membrane that is a replica of the gel (also known as a Southern blot). Because of its specificity, the radioactive probe can be used to demonstrate genotypic similarities between individuals. In DNA fin- gerprinting, the relative positions of radiolabeled bands in a gel are determined by the size of the DNA fragments in each band. The size of the fragments reflect variations in individuals’ DNA. The evidence needed for DNA fingerprinting can be obtained from any biological material that contains DNA: body tissues, body fluids (blood and semen), hair follicles, etc. DNA analysis can even be done from dried material, such as blood stains or mummified tissue. If a sample of DNA is too small it may be amplified using PCR techniques. The DNA is then treated with restriction enzymes that cut the DNA into fragments of various length. Restriction Digestion of DNA Because they cut DNA, restriction enzymes are the “chemical scissors” of the molecular biologist. When a particular restriction enzyme “recognizes” a particular recognition sequence (four- or six- base pair (bp)) on a segment of DNA, it cuts the DNA molecule at that point. The recognition sequences for two commonly used enzymes, Eco RI and Pst I , are shown below. The place on the DNA backbones where the DNA is actually cut is shown with ( % ) symbol: For the enzyme Eco RI For the enzyme Pst I Like all enzymes, restriction enzymes function best under specific buffer and temperature condi- tions. The proper restriction enzyme buffer has been included with the DNA sample in this kit, so that when the rehydrated DNA and enzymes are mixed, the ideal conditions are created for the enzymes to function optimally. The final reaction buffer consists of 50 mM Tris, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DDT, pH 8.0, which is the ideal condition for Eco RI and Pst I enzymes to function. G A A T T C C T T A A G C T G C A G G A C G T C % % % %

DNA Sample Movement of DNA Fragments Anode Cathode Power Supply (120V) Agarose gel Figure 1 Figure 2 Equipment for Electrophoresis Edge ® Electophoresis Apparatus Microcentrifuge Micropipet Block Heater

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Making DNA Visible DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. A sample loading buffer containing two bluish dyes is added to the DNA samples. The loading dye does not stain the DNA itself but makes it easier to load the samples and monitor the progress of the DNA elec- trophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. Bromophenol blue, the “faster” dye, comigrates with DNA fragments of approximately 500 bp in a 1% agarose gel, while the xylene cyanol, the “slower” dye, comigrates with DNA fragments of approxi- mately 4,000 bp in a 1% agarose gel. Staining the DNA pinpoints its location on the gel. A fluorescent dye (SYBR ® ) is incorporated in the agarose gel and will attach to the DNA trapped in the gel. The DNA bands stained with SYBR ® are colorless in visible light but fluoresce orange when viewed under UV light. When the bands are visible under a UV light, you can compare the DNA restriction patterns of the different samples of DNA. The standard fluorescent stain normally used is ethidium bromide. A major disadvantage of using ethidium bromide is that it is a toxic mutagen and must be used with caution. The SYBR ® stain that you will be using is fairly non-toxic. However it is always standard practice to wear gloves when working with DNA. Reliability of DNA Evidence Two major factors affecting the reliability of DNA fingerprinting technology in forensics are population genetics and genetic statistics. In humans there are thousands of RFLP loci or DNA segments that can be selected and used for fingerprinting analysis. Depending on demographic factors such as ethnicity or geographic isolation, some segments will show more variation than others. In gen- eral one can assume that any two humans are 99.9% identical DNA sequence. Thus they will differ by only 1 base pair (bp) in 1,000. It is necessary to examine areas that differ to create a useful DNA finger- print. Some populations show much less variation in particular DNA segments than others. The degree of variation will affect the statistical odds of more than one individual having the same sequence. If 90% of a given population has the same frequency in its DNA fingerprinting pattern for a certain DNA segment, then very little information will be attained. But if the frequency of a DNA pattern turning up in a population for a particular segment is extremely low, then this segment can serve as a powerful tool to discriminate between individuals in that population. Different populations show different patterns in their genotypes due to the contributions made to their individual gene pools over time. Therefore, in analyzing how incriminating the DNA evidence is, one needs to ask the question: “Statistically, how many people in a population have the same pattern as that taken from a crime scene: 1 in 1,000,000? 1 in 10,000? Or, 1 in 10?”

Procedure Part 1 - Restriction Digestion of DNA Samples Upon careful observation, it is apparent that the only difference between the DNA of different indi- viduals is the linear sequence of their base pairs. In the lab, your team will be given 6 DNA samples. Recall that your task is to determine if any of them came from the same individual or if they came from different individuals. Thus far you have learned the following: • The similarities and differences between the DNA from different individuals. • How restriction endonucleases cut (hydrolyze) DNA molecules. • How adding the same restriction endonuclease to two samples of DNA might provide some clues about differences in their linear base pair sequence. Now that you have a fairly clear understanding of these three items you are ready to proceed to the first phase of the DNA fingerprinting procedure—performing a restriction enzyme digest of your DNA samples. Step 1: Combine and React. Adjust your micropipet to read 10 μ l and add 10 μ l of the enzyme mix “ENZ” to each reaction tube as shown below. The DNA sample is already in the tube. You do not need to change pipet tips for this step. Note: Change tips whenever you switch reagents, or, if the tip touches any of the liquid in one of the tubes accidentally. When in doubt, change the tip! When working with DNA samples gloves should always be used to avoid contamination of samples with your own DNA

Now your DNA samples should contain: Total DNA Samples Eco RI/ Ps tI Reaction (10 μ l each) Enzyme Mix Volume Crime Scene [CS] 10 μ l 20 μ l Suspect 1 [S1] 10 μ l 20 μ l Suspect 2 [S2] 10 μ l 20 μ l Suspect 3 [S3] 10 μ l 20 μ l Suspect 4 [S4] 10 μ l 20 μ l Suspect 5 [S5] 10 μ l 20 μ l Step 2: Mix the tube contents. Tightly cap each tube. Mix the components by gently flicking the tubes with your finger. Pulse the tubes in the centrifuge for 15 seconds to force the liquid into the bottom of the tube to mix and com- bine reactants. Be sure the tubes are in a BALANCED arrangement in the rotor. Step 3: Incubate the samples. Place the tubes in the metal heat block and incubate them at 37°C for 20-25 minutes . Make sure to tightly cap each tube to prevent evaporation during incubation. After the incubation, place the tubes in the foam holder and return to your bench area. Step 4: Micropipet Practice ( this may have been done in your previous lab session ) Note : While you are waiting for the incubation, you will practice filling wells in a gel using a colored dye solution. It is critical that you are proficient in using a micropipet to fill the gel wells with DNA samples. You will only have enough of your actual digested DNA samples to fill only one well each. If you mess up adding your sample you will not get another chance. 1. Obtain a practice gel from the instructor. This gel will have 4 rows of wells. Place the gel in the electrophoresis apparatus and add the practice buffer solution until it completely covers the gel. Adjust the micropipet to deliver 20 m l. Each member of your group is to practice filling an entire row of wells with 20 m l of the practice blue dye solution in each well. You do not have to change micropipet tips during this practice. Flick Tap

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2. Place the tip of the micropipet just inside the well and very slowly add the practice dye solution. Make sure the tip of the micropipet does not pierce the gel at the bottom of the well or that the pipet tip is not too high above the well. Do not release the micropipetor plunger until you remove the tip out of the buffer or you will suck your sample back out. If you do this cor- rectly all 6 wells across the gel should appear as consistently dark blue rectangles. 3. When you are done, pour the used buffer solution and practice gel into the waste container. Part 2 - Agarose Gel Electrophoresis Step 1: Agarose Gel and Electrophoresis Apparatus Obtain a pre-made agarose gel and carefully remove the well comb and rubber end caps from the casting tray. Be careful not to rip the gel as you remove the comb and rubber end caps. The agarose gel contains SYBR ® DNA stain. This stain will attach to the DNA fragments and will fluoresce under UV light. Place casting tray in the Edge ® electrophoresis apparatus and fill with fresh buffer solution. The buffer solution is premeasured. Add all of it to the apparatus. The gel must be completely covered with the buffer solution. Your wells need to be on the cathode side (–) of the apparatus. Proper micropipet tip position for filling wells with samples Cross section of gel wells No No Yes Anode (+ red) Cathode (– black) Position wells near cathode Agarose Gel

Step 2: Loading Samples a. Adjust the micropipet to deliver 5 m l . Add 5 μ l of sample loading dye “LD” to each of your incubated tubes. You do not need to change tips for this step. DNA Samples Loading dye Crime Scene [CS] 5 μ l Suspect 1 [S1] 5 μ l Suspect 2 [S2] 5 μ l Suspect 3 [S3] 5 μ l Suspect 4 [S4] 5 μ l Suspect 5 [S5] 5 μ l b. Tightly cap each tube. Mix the components by gently flicking the tubes with your finger. Pulse spin the tubes in the centrifuge for 15 seconds to bring the contents to the bottom of the tube. c. Obtain a tube of DNA size marker . The loading dye will already be in the tube. d. Adjust your micropipet to read 20 μ l. Using a separate pipet tip for each sample , load your digested DNA samples into the gel wells. Load from left to right (DNA size marker in lane number one). Lane 1: DNA size marker, clear tube, 20 μ l Lane 2: CS, green tube, 20 μ l Lane 3: S1, blue tube, 20 μ l Lane 4: S2, orange tube, 20 μ l Lane 5: S3, violet tube, 20 μ l Lane 6: S4, red tube, 20 μ l Lane 7: S5, yellow tube, 20 μ l

e. Close the lid on the gel box. f. Set the voltage to 100 volts and run the samples for at least 35 - 40 min. Bubbles forming at the electrodes indicates that current is running through the buffer and gel. g. When the electrophoresis is complete, hit the stop button. h. To view your gel push the blue visualization paddle. The lab lights may need to be turned down for best viewing. i. Take a photo of your gel with your phone and compare your gel to the photo provided for constructing your standard curve. Pour the used buffer and your finished gel into the provided waste container. Your empty microcentrifuge tubes can be discarded in the trash. Since DNA is naturally colorless, it is not immediately visible in the gel. Unaided visual exami- nation of the gel after electrophoresis indicates only the positions of the loading dyes and not the positions of the DNA fragments. The DNA fragments will pick up the SRYB ® stain as they move down the gel and will be visible when the visualization paddle is depressed. For fingerprinting analysis, the following information is important to remember: • Each lane has a different sample of DNA • Each DNA sample was treated with the same restriction endonucleases. Electrophoresis of DNA Size Markers of known base pair values 10,000 bp 8000 bp 4000 bp 3000 bp 2000 bp 1500 bp 1400 bp 1000 bp 750 bp 500 bp 400 bp 300 bp 200 bp

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Part 3 - Record and Analyze Results Quantitative Analysis of DNA Fragment Sizes ( you can do this analysis while your gel is running ) If you were on trial or were trying to identify an endangered species, would you want to rely on a technician’s eyeball estimate of a match, or would you want some more accurate measurement? In order to make the most accurate comparison between the crime scene DNA and the suspect DNA, other than just a visual match, a quantitative measurement of the fragment sizes needs to be com- pleted. Use the provided photo of a typical results to make these measurements. You can do these calculations while you are waiting for your own gel to run. If your gel works properly your band pattern should look very similar to the band pattern in the photo. a. Using a ruler, measure the distance (in mm) that each of your DNA size marker fragments or bands traveled from the well (lane 1). Measure the distance from the bottom of the well to the center of each DNA size marker band and record your values in the data table on the next page. The data in the table will be used to construct a standard curve and to estimate the sizes of the crime scene and suspect restriction fragments. b. To make an accurate estimate of the fragment sizes for either the crime scene or suspect DNA samples, a standard curve is created using the distance (x-axis) and fragment size (y-axis) data from the known DNA size marker. Using a lab iMac ® open the DNA lab graphing file (found on the desktop) and enter your measured distances in the table for each of your DNA size marker bands. The graphing program will automatically generate a standard curve. Print out a copy for each member of your lab team (include with your final report) See sample on next page. c. To estimate the size of an unknown crime scene or suspect fragment, measure the distance that fragment traveled (in mm). Locate that distance on the x-axis of your standard graph. From that position on the x-axis, read up to the standard line, and then follow the graph line to over to the y-axis. You might want to draw a light pencil mark from the x-axis up to the standard curve and over to the y-axis showing what you’ve done. Where the graph line meets the y-axis, this is the approximate size of your unknown DNA fragment. Do this for all crime scene and suspect fragments. See sample on next page. d. Compare the fragment sizes of the suspects and the crime scene. Is there a suspect that matches the crime scene? How sure are you that this is a match?