Download Standards for the English Language Arts - SCHS

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.