This lab is designed to familiarize you with gel electrophoresis and how it is used to separate the DNA fragments that result from a restriction endonuclease digest. You will also learn how to use graphing and a “marker” (DNA fragments of known size), to determine the sizes of the DNA fragments of unknown size. In this lab, the marker is the lambda DNA cut with HindIII, for which fragment sizes are given in the table in the Analysis section of this guide. The DNA loaded onto the gel in this lab is from lambda, a bacteriophage (also known as a bacteria virus) that was used in early studies of gene regulation. Each sample tube contains the entire genome of lambda, only 48,502 base pairs in length! Two of the samples have been cut with restriction endonucleases. Restriction endonucleases (also called restriction enzymes) are proteins that cleave DNA at specific sequences. Restriction enzymes were originally isolated from bacteria. In bacteria they protect the cell by cleaving DNA of any invading virus. The bacteria’s own DNA is protected from cutting by being modified in a way that interferes with the restriction enzyme. For example, the bacterial DNA may have a methyl group added to the DNA sequence a specific enzyme would normally cut. The methyl group prevents the enzyme from functioning. Restriction enzymes find their cutting sites by recognizing a short sequence of nucleotides called a recognition

sequence. The recognition sequence may include the site where the restriction enzyme cuts or it may serve as

the sequence the enzyme needs to recognize in order to cut a nearby sequence. When a DNA molecule is cut

by restriction enzymes, the result will always be a set of restriction fragments, which will have at least one

single-stranded end, called a sticky end.

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In this lab activity, you will load three different samples of lambda DNA onto a gel. One DNA sample is cut with the restriction enzyme EcoRI, one is cut with the restriction enzyme HindIII, and one is the control, which is uncut. The DNA samples are loaded into the wells of an agarose gel and electrophoresed. Because DNA is negatively charged, an electrical field applied across the gel causes the DNA fragments in the samples to move from the origins (the wells) through the gel matrix toward the positive electrode (the anode). Smaller DNA fragments migrate faster than larger ones, so restriction fragments of differing sizes become concentrated into distinct bands during electrophoresis.

Electrophoresis can be performed with gels containing different percentages of agarose. A 0.8% gel (0.8g of agarose per 100 mL of buffer) is used in this lab because it is the best option for separating the size range of DNA fragments in these samples. The table below shows what percentage gels are typically used to separate different size fragments. Keep in mind that the pores in lower-percentage gels are larger and therefore allow fragments of all sizes to move more easily through the matrix of the gel. The pores in higher-percentage gels are smaller, making it more difficult for DNA fragments, especially larger ones, to move through the gel.

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Pre-Laboratory Questions 1. What do restriction enzymes do? Describe how a restriction enzyme can be used to determine if there is a

single base-pair change between two otherwise identical pieces of DNA.

2. Why do DNA fragments migrate toward the positive end of a gel electrophoresis chamber when electricity is applied?

3. In general, what percentage gel do you think would separate large fragments of DNA more effectively – a gel with a low percentage of agarose, or a gel with a high percentage of agarose? Explain your answer.

4. You read a poster from a student electrophoresis project comparing the effectiveness of two different concentrations of agarose gels at separating specific DNA fragments. In the experiment, the students ran the two different gels side by side at 120 volts. One gel ran for 30 minutes, and the other ran for 45 minutes. Do you think the group’s results are valid? Why or why not?

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Procedure: Loading the Gel & Electrophoresis Load the contents of each supplied tube (lambda/EcoRI, lambda/HindIII, and uncut lambda) into a separate well in the gel. Be sure to record the order that you load the samples in. Use a fresh loading tip for each tube. You teacher will provide you with instruction on how to use the micro pipets for loading the wells. Here are some general techniques:

1. Before loading your gel with samples of DNA, you should practice using the pipette or other loading device. Keep practicing until you feel comfortable loading and expelling a sample.

2. Make sure you record the order in which you load the samples. Be sure to use a fresh loading device (either plastic micropipette or other type of pipette) for each sample.

3. Be sure you know how to use the pipette properly. When in doubt, ask your teacher. Take care not to puncture the bottom of the well with the pipette tip when you load your samples.

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Analysis & Questions Over the years, understanding of the details of how electrophoresis works has become increasingly complex. A great deal has been learned about how the DNA molecules being separated interact with the matrix (in this case, agarose), how molecules move through the agarose, and how the agarose matrix itself is influenced by the electric field. Mathematical formulas have been developed for describing the relationship between the molecular weight of a DNA fragment and its mobility (i.e., how far it runs on the gel.) In general, linear, double-stranded DNA fragments like the ones used in this lab migrate at rates inversely proportional to the log10 of their molecular weights. Because of this relationship, the molecular weight of a DNA fragment can be interpolated from the distance the DNA fragment moves through a gel. In order to interpolate the molecular weight, a DNA standard, composed of DNA fragments of known molecular weight, is run on the same gel as the unknown fragments and is used to create a standard curve. The standard curve, in this case a straight line, is created by graphing the relative mobility (Rf) of each DNA fragment versus the log10 of its molecular weight. Using Rf instead of a simple measure of how far a DNA fragment has run from the well creates a standard curve that is closer to a straight line and thus yields a more accurate estimation of molecular weight. For this exercise we will calculate Rf as the ratio of the distance that the given DNA band travels into the gel over the distance that the loading dye travels into the gel.

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Big Idea Assessment Use the figure to the right to answer the following questions. The DNA profiles below represent four different individuals. 1. Which of the following statements is consistent with the results?

a. B is the child of A and C. b. C is the child of A and B. c. D is the child of B and C. d. A is the child of B and C.

2. Which of the following statements is most likely true? a. D is the child of A and C. b. D is the child of A and B. c. D is the child of B and C. d. A is the child of C and D.

3. Both members of a couple come from families with a history of a severe disease (i.e., affected children do

not live beyond a year or two after birth) caused by an autosomal recessive mutation. The disease appears in all people homozygous recessive for the mutation and is present from birth on. People heterozygous for the mutation have, as they get older and to varying degrees, a higher risk for specific health problems that require medical care. The couple is deciding whether, before having children, they should be genetically tested for the mutation. List the pros and cons of their being genetically tested. Consider the question from all perspectives (with respect to privacy, medical care, emotional effects, and ethical implications). Address each of these in your pros/cons list.

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Application Practice Cystic fibrosis is an autosomal recessive genetic disease that occurs in the US with an approximate frequency of 1 in 3500 births. The disease results from mutation of the cystic fibrosis transmembrane conductor gene (CFTR) which codes for a protein (CFTR protein) that regulates the flow of chloride ions across membranes. As of 2012, at least 1906 different mutations had been identified in the CFTR gene. Different mutations confer different degrees and sometimes different types of disease. Some of the mutations do not cause disease at all. Typically, the disease is characterized by thickened secretions throughout the body, which most notably affect the lungs, pancreas, and hepatobiliary system (liver, gallbladder, and bile ducts). The thickened secretions in the lungs lead to difficulty in clearing mucus from the lungs, thus increasing the chance of infection. Thickened secretions in the pancreas affect the distribution of vital digestive enzymes.

A sweat chloride test (which measures the amount of chloride ions in a person’s sweat – most CF patients have elevated levels) is the classic diagnostic test for this disease, but it is not always definitive. In cases where neither the sweat chloride test nor symptoms allow for definitive diagnosis, genetic testing can help. Review the simulated genetic screen scenario on the following pages. Use the information found in the scenario and the ideal gel image to answer the questions that follow.

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Review the ideal gel example below displaying the results you obtained from the electrophoresis. Use the ideal gel to answer the following questions.

1. Explain how electrophoresis can be used as a part of genetic testing.

2. Why do you think it would be necessary to use PCR to amplify the region of DNA that you wish to analyze?

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3. Identify three controls included in the simulated genetic screen and explain why they are necessary.

4. Compare the DNA fingerprints produced by restriction digestion of the PCR products generated from the children’s DNA and the DNA fingerprints created by digestion of the PCR products from the control CF mutant and wild-type DNA.

a. Which pattern does the DNA fingerprint from each child’s DNA match – the pattern from the mutant DNA, the pattern from the wild-type DNA, or a combination of both?

b. What does this tell you about the genotype of the children with respect to this CF mutation?

5. Remember that this particular mutation in the cystic fibrosis gene destroys a restriction enzyme site. If you did not know about the destroyed restriction site, what about the fragment patterns on your gel would indicate to you that a restriction enzyme site had been destroyed?

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Creating Restriction Plasmid Maps Refer to the following web page for help with creating plasmid maps: http://www.carolina.com/pdf/activities-articles/plasmid-mapping-exercises-2008.pdf . Students may also refer to their virtual gel electrophoresis lab for help with this concept. The following animations may also be useful. Students are HIGHLY encouraged to watch these before they attempt the practice problems on the following pages. https://www.youtube.com/watch?v=v2T8Y3-8674 https://www.youtube.com/watch?v=8FqMUF96cPE Restriction enzymes are proteins that separate a DNA molecule at a specific location (locus). Think of them as molecular scissors. The terms "cut," "digest," or "restrict" may be used to describe the action of a restriction enzyme. Whenever a DNA molecule is cut with a restriction enzyme, the resulting pieces often need to be reassembled in a map representing the relative locus where the restriction enzyme cut the DNA molecule. This is because scientists are usually trying to determine where a specific gene is located in a certain piece of DNA. They start by using restriction enzymes that act close to either end of the gene of interest. Once the gene has been located on a piece of DNA, it is often useful to determine where the piece of DNA was originally located. To do this, scientists try to construct a map of the original piece of DNA using their experimental data. Because plasmids are rings or circles of DNA, a restriction enzyme that cuts a plasmid once results in a linear piece of DNA that has the same number of base pairs as the original plasmid. A restriction enzyme that cuts a plasmid twice results in 2 linear pieces of DNA whose total number of base pairs equals the number of base pairs in the original plasmid. When 2 restriction enzymes cut the same plasmid, it is referred to as a double digest. It is usually necessary to use at least 2 restriction enzymes to map a plasmid. However, it is not uncommon for as many as 6 or 8 restriction enzymes to be used. Plasmid maps normally take the form of a circle. The name of the restriction enzyme and the relative locus where the enzyme cuts the plasmid are shown on the map. The center of the map is labeled with the total number of base pairs in the plasmid. Mapping a plasmid is basically a game of logic. The key is to remember to account for all experimental data. Think of it as taking a clock apart and putting it back together again with no parts remaining. Here is an example of a gel and the corresponding plasmid map:

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