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Lesson 5.2: Molecular Biological Techniques for Diagnosing Disease Preface Molecular techniques are changing the way diseases are diagnosed and drugs are prescribed. Since the mid-1990’s DNA analysis has changed how the courts and juries determine the guilt or innocence of criminal suspects. The popularity of television programs that detail numerous forensic crime-solving techniques has exposed many students to the results of molecular biological techniques. For scientific literacy, it is important that students gain an understanding of how these various techniques are performed and their limitations. The polymerase chain reaction, developed by Kary Mullis in 1983, allowed genetic analysis to be performed on minute quantities of DNA—even as small an amount as the DNA from a single cell. In only 30 cycles of PCR, it is possible to produce over a billion copies of a section of DNA derived from a single cell. This amplification process allowed reliable genetic analysis of crime scenes where single drops of blood, semen, or sweat can now pin-point the perpetrator. PCR also allowed for the non-invasive evaluation and screening of patient DNA for genetic abnormalities. New, less invasive techniques for screening fetal DNA are the direct result of the amplification capabilities of PCR. PCR is a very elegant and simple procedure that mimics the DNA replication process found in cells. The key was the isolation of the polymerase enzyme which enabled DNA replication to occur in vitro with any DNA template. Mullis originally used a series of water baths at different temperatures to complete the various steps required for one DNA replication cycle: DNA denaturation, attachment of primers, and elongation of the DNA copy. Because the polymerase enzyme he used was destroyed by the heat of the denaturation process, fresh enzyme had to be added for each cycle. Key advances that led to the rapid adoption of the PCR technique were the isolation of a heat stable polymerase enzyme from a thermophilic bacteria and the invention of a thermocycler to rapidly and automatically adjust the temperature for each step of the cycle. The heat stable Taq polymerase eliminated the need to add fresh enzyme for each cycle. The thermocycler allowed the process to be completely automated. The Human Genome Project completed in 2002 stimulated the targeted analysis of the DNA from individuals with specific diseases to determine which gene or genes were linked to the disease or to disease susceptibility. Numerous genetic links have been established to a wide variety of diseases and disorders including heart disease, neuromuscular disorders, dementia, schizophrenia, and cancer. Familial hypercholesterolemia leads to early onset of heart disease resulting in premature death. The most common cause is a single genetic mutation to the LDL receptor that is referred to as the FH mutation. A person homozygous for the mutation can only produce the defective LDL receptor, resulting in the accumulation of high levels of LDL in the blood. Often people homozygous for the FH mutation die of heart disease in their 20’s. A person who is heterozygous for the FH mutation produces both the normal and the defective LDL receptor. Because this person’s cells have some normal receptors, the cells are capable of binding LDL, and the blood levels of LDL are not as high as in the homozygous condition. However, the blood levels of LDL are still above normal and heterozygous individuals often die in their 40’s or 50’s of heart disease. In this lesson, students will perform gel electrophoresis on mock DNA samples in order to diagnose the genetic disease familial hypercholesterolemia. In preparation for the analysis of the DNA samples, the students will calculate the amount of amplification of DNA produced by the polymerase chain reaction (PCR) and watch animations of how PCR and restriction fragment length polymorphism (RFLP) analysis are used to diagnose disease. Concepts 1. DNA from numerous sources including blood and saliva can be amplified and analyzed. 2. The Polymerase Chain Reaction (PCR) exponentially increases the number of DNA molecules. 3. Restriction Fragment Length Polymorphism allows for genetic diseases and disorders to be diagnosed by analysis of DNA samples without DNA sequencing. 4. DNA gel electrophoresis separates DNA fragments based on size and is used in Restriction Fragment Length Polymorphism analysis. Standards and Benchmarks Addressed National Science Education Standards Unifying Concepts and Processes: As a result of activities in grades K-12, all students should develop understanding and abilities aligned with the following concepts and processes: Change, constancy, and measurement Form and function Standard A: Science As Inquiry: As a result of activities in grades 9-12, all students will develop: Abilities necessary to do scientific inquiry Understandings about scientific inquiry Standard C: Life Science: As a result of activities in grades 9-12, all students should develop an understanding of: Molecular basis of heredity Standard E: Science and Technology As a result of activities in grades 9-12, all students should develop: Understandings about science and technology Principles and Standards for School Mathematics Number Operations: Instructional programs from pre-kindergarten through grade 12 should enable all students to understand: Numbers, ways of representing numbers, relationships among numbers, and number systems. Compute fluently and make reasonable estimates. Algebra: Instructional programs from pre-kindergarten through grade 12 should enable all students to: Understand patterns, relations, and functions. Measurement: Instructional programs from pre-kindergarten through grade 12 should enable all students to: Understand measurable attributes of objects and the units, systems, and processes of measurement. Apply appropriate techniques, tools, and formulas to determine measurements. Problem-Solving Instructional programs from pre-kindergarten through grade 12 should enable all students to: Solve problems that arise in mathematics and in other contexts. Monitor and reflect on the process of mathematical problem-solving. Communication Instructional programs from pre-kindergarten through grade 12 should enable all students to: Communicate their mathematical thinking coherently and clearly to peers, teachers, and others. Use the language of mathematics to express mathematical ideas precisely. Connections Instructional programs from pre-kindergarten through grade 12 should enable all students to: Recognize and apply mathematics in contexts outside of mathematics. National Healthcare Cluster Foundation Standards Academic Foundation Healthcare professionals will know the academic subject matter required for proficiency within their area. They will Accountability Criteria 1.1 use this knowledge as needed in their role. Human Structure and Function Classify the basic structural and functional organization of the human body including chemical, cellular, tissue, organ, and system. Analyze the interdependence of the basic structures and functions of the human body as they relate to wellness, disease, disorders, therapies, and care/rehabilitation. Accountability Criteria 1.2 Diseases and Disorders Compare selected diseases/disorders including respective classification(s), causes, pathogenesis, diagnoses, therapies, and care/rehabilitation. Communications Healthcare professionals will know the various methods of giving and obtaining information. They will communicate effectively, both orally and in writing. Concepts of Effective Communication Use medical terminology to communicate information including data and observations. Accountability Criteria 2.1 Accountability Criteria 2.3 Written Communication Skills Organize technical information and summaries. Employability Skills Healthcare professionals will understand how employability skills enhance their employment opportunities and job satisfaction. They will demonstrate key employability skills and will maintain and upgrade skills, as needed. Career Decision-making Recognize levels of education, credentialing requirements, employment opportunities, workplace environments, and career growth potential for a service area. Accountability Criteria 4.3 Accountability Criteria 4.4 Employability Preparation Execute work assignments and formulate solutions to problems using critical thinking skills. Information Technology Applications Accountability Criteria 11.2 Healthcare professionals will use information technology applications required within all career specialties. They will demonstrate use as appropriate to healthcare applications. Information Technology Implement communications using technology (i.e. Fax, E-mail, and Internet) to access and distribute data and other information. Execute the use of software, hardware, and the Internet Standards for the English Language Arts Standard 5: Standard 8: Standard 12: Students employ a wide range of strategies as they write and use different writing process elements appropriately to communicate with different audiences for a variety of purposes. Students use a variety of technological and informational resources (e.g. libraries, databases, computer networks, video) to gather and synthesize information and to create and communicate knowledge. Students use spoken, written, and visual language to accomplish their own purposes (e.g. for learning, enjoyment, persuasion, and the exchange of information). Standards for Technological Literacy Standard 3 BM J Standard 12 BM P Standard 14 BM M Students will develop an understanding of the relationships among technologies and the connections between technology and other fields of study. Technological progress promotes the advancement of science and mathematics. Likewise, progress in science and mathematics leads to advances in technology. Students will develop the abilities to use and maintain technological products and systems Use computers and calculators to access, retrieve, organize, process, maintain, interpret, and evaluate data and information in order to communicate. Students will develop an understanding of and be able to select and use medical technologies. The sciences of biochemistry and molecular biology have made it possible to manipulate the genetic information found in living creatures. Performance Objectives It is expected that students will: Calculate the amplification of DNA during the polymerase chain reaction. Create a graph of the amplification rate. Use proper laboratory techniques to separate DNA fragments by gel electrophoresis. Analyze the results of the gel electrophoresis to correctly diagnose the presence of the familial hypercholesterolemia mutation. Assessment Explanation 1. Students will describe how the polymerase chain reaction amplifies DNA. 2. Students will explain how gel electrophoresis separates DNA fragments based on size. 3. Students will explain how polarity, voltage, time, and agarose concentration affect the results of gel electrophoresis. Application 1. Students will analyze DNA fragments for Restriction Fragment Length Polymorphism to correctly diagnose the presence of a mutation. Essential Questions 1. How do crime scene investigators get enough DNA evidence from a single drop of blood? 2. What is PCR? 3. How is DNA analyzed without sequencing it? 4. What does PCR do and how does it work? 5. Can genetic diseases or disorders be diagnosed using a small blood or saliva sample from a patient? 6. Why are DNA tests on television programs and movies shown as patterns of stripes or bands on film or in gels? 7. What is gel electrophoresis and how are the results interpreted? Key Terms Agarose A polysaccharide obtained from seaweed that is used as the supporting medium in gel electrophoresis. Allele Alternative versions of a gene that produce distinguishable phenotypic effects. Amplification A usually massive replication of genetic material and especially of a gene or DNA sequence. Electrophoresis The movement of suspended particles through a fluid or gel under the action of an electromotive force applied to electrodes in contact with the suspension. Exponential Rapidly becoming greater in size. In mathematics used to describe a mathematical entity such as a curve, function, equation, or series that contains, is expressed as, or involves numbers or quantities raised to an exponent. Familial A metabolic disorder that is caused by defective or absent Hypercholesterolemia receptors for LDLs on cell surfaces, that is marked by an increase in blood plasma LDLs and by an accumulation of LDLs in the body resulting in an increased risk of heart attack and coronary heart disease, and that is inherited as an autosomal dominant trait. Heterozygous Having two different alleles for a given gene. Homozygous Linear Mutation Restriction Endonuclease PCR (Polymerase Chain Reaction) Phenotype Polymerase Polymorphism Having two identical alleles for a given gene. Relating to a straight line or capable of being represented by a straight line. A rare change in the DNA of a gene, ultimately creating genetic diversity. A degradative enzyme that recognizes specific nucleotide sequences and cuts up DNA. A laboratory technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides. The physical and physiological traits of an organism that are determined by its genetic makeup. Any of several enzymes that catalyze the formation of DNA. The coexistence of two or more distinct forms in the same population. Day-by-Day Plans Time: 5 days Note: The teacher will need a total of approximately 2 hours preparation time prior to students starting Activity 5.2.2. See Unit 5 Teacher Notes. Day 1 - 2: The teacher reviews the Unit 5 Teacher Notes, the Unit 5 Key Terms crossword puzzle, and its solution. The teacher asks the students if they can recall a scene from a movie or a television show where investigators used DNA to convict a criminal suspect. The teacher leads the students in a discussion of various sources of DNA and how the information is used. Discussion should lead students to recall that DNA can be obtained from a wide variety of sources including: blood drops, mouth swabs, skin collected from under fingernails, envelopes or stamps licked by someone, and sweat from the headband of a hat. The point is for students to recall that DNA can be collected from minute quantities of any body fluid that contains cells. The teacher asks Essential Question 1. Students may be able to answer the Essential Question and respond saying “PCR” or even “Polymerase Chain Reaction.” The teacher asks Essential Questions 2 and 3. The teacher asks “What do you know about PCR?” or “What do you see on the television shows or movies when the investigators do PCR?” Students respond and describe what they have seen. The teacher writes the responses on the board or uses the Rapid Fire feature of Inspiration. This establishes what students know about DNA analysis and PCR. The teacher introduces and reviews the definitions of the Key Terms. The teacher asks Essential Question 4 and introduces Activity 5.2.1: How Does PCR Amplify DNA? Students complete Activity 5.2.1. The teacher monitors and checks student performance as they complete the activity. Specific teacher checkpoints are written into the activity; at these spots the students require teacher verification before proceeding with the activity. The teacher uses the Activity 5.2.1 Answer Key to assess student performance. The teacher reviews the student responses to the Conclusion Questions and assesses student performance and understanding of the concepts. It is important that the students understand the differences between a linear and an exponential relationship. The teacher should also assess student understanding of the function of each step in the PCR cycle and that the selectivity of the PCR reaction is due to the selection of the primers. Day 3 – 5: The teacher tells the story of a family with a history of early death due to heart problems or strokes. The story could be similar to this one. There was a Finnish family whose two sons died of heart attacks before they reached the age of 25. The mother died at age 47 of a heart attack and the maternal grandmother had a stroke at age 48 and a maternal uncle died of a heart attack at age 55. The paternal grandfather died of a stroke at age 52. The teacher asks the students to create a pedigree for the fictional family. The teacher asks, “What could be the cause of so many heart attacks and strokes in this family?” The students respond with possible causes. The teacher lists the causes the students suggest on the board or uses the Rapid Fire feature of Inspiration. If the students have not suggested high cholesterol, then the teacher should guide them to make that suggestion. The teacher insures through the discussion that students realize there is some factor in the genetics of this family that is responsible to the high incidence of heart disease and stroke. The teacher asks Essential Questions 5 - 7. The teacher introduces Activity 5.2.2: What Is Familial Hypercholesterolemia and How Is It Diagnosed? The teacher demonstrates how to effectively and safely use the micropipettor and agarose gel electrophoresis apparatus. The teacher demonstrates how to load a DNA sample into a well of an agarose gel. The students complete Activity 5.2.2. The teacher monitors student performance and insures that each electrophoresis apparatus is safely and correctly connected to the power supply. The teacher instructs students on how the gels will be stained and viewed after the electrophoresis is stopped. The teacher reviews the student responses to the Conclusion Questions and assesses student understanding of the concepts related to restriction enzyme analysis and gel electrophoresis. The teacher should also insure that students understand that when a patient is heterozygous for some genetic conditions, that both the normal and the abnormal proteins will be produced, and this may lessen the effects of a disease condition. This is the case with both the familial hypercholesterolemia and the sickle cell mutations. Students complete Activity 5.2.3: Careers That Use Molecular Biology. Instructional Resources Word Documents Activity 5.2.1 How Does PCR Amplify DNA? Activity 5.2.2 What is Familial Hypercholesterolemia How Is It Diagnosed Activity 5.2.3 Careers That Use Molecular Biology Unit 5 Crossword Puzzle Unit 5 Crossword Puzzle Solution Answer Keys and Assessment Rubrics Activity 5.2.1 Answer Key Teacher Guidelines Unit 5: Teacher Notes Reference Sources Campbell, A. Malcom. 2002. Davidson College. How to Use a Micropipettor. Accessed May 31, 2008. Available at: http://www.bio.davidson.edu/COURSES/Molbio/Protocols/pipette.html. Campbell, Neil and Reece, Jane. 2005. Biology. 7th edition. Pearson Education, Inc., publishing as Benjamin Cummings. San Francisco. Cold Springs Harbor Laboratory. 2003. Manipulation. Accessed May 31, 2008. Available at http://www.dnai.org/b/index.html. Max Animations. Genetics. PCR Animation. Accessed May 31, 2008. Available at: http://www.maxanim.com/genetics/PCR/PCR.htm. Rutgers University. PCR Animation. Accessed May 31, 2008. Available at: http://spine.rutgers.edu/cellbio/flash/pcr.htm. Thinkwell’s Electrophoresis. Separating DNA. Accessed May 31, 2008. Available at http://207.207.4.198/pub/flash/4/4.html.