Download DNA Background

DNA Extraction Lab
Instruction Sheet
DNA is in all living things. Single molecules of DNA are long and stringy. Each cell of your body
contains six feet of DNA, but it’s only one-millionth of an inch wide. To fit all of this DNA in
your cells, it needs to be packed efficiently. To solve this problem, DNA twists tightly and
clumps together inside cells. Even when you extract DNA from cells, it still clumps together,
though not as much as it would inside the cell. The human body contains about 100 trillion
cells, each of which contains six feet of DNA. This means that our bodies contain more than a
billion miles of DNA! In this lab we will extract DNA from split peas.
Procedure: (Teacher Part)
1. In class, your teacher will place 400 mL of split peas, a large pinch of table salt, and
800 mL of cold water into a blender and blend on high for 30 seconds. The blender
separates the pea cells from each other, so you now have a real thin pea-cell soup.
2. Your teacher will then pour the pea mixture through a strainer into a beaker.
3. Your teacher will divide the mixture equally into test tubes.
Procedure: (Student Part)
4. Add 2 drops of detergent to the pea mixture and agitate solution for 1 minute.
- allow to sit for 5-10 minutes
5. Add a pinch of enzymes (meat tenderizer) to each test tube and SLOWLY mix the solution.
BE CAREFUL! IF YOU STIR TOO HARD, YOU’LL BREAK UP THE DNA, MAKING IT HARDER TO SEE.
6. Tilt your test tube and using a pipette, slowly add rubbing alcohol into the tube down the
side so that it forms a layer on top of the pea mixture. Pour until you have about the same
amount of alcohol in the tube as pea mixture.
7. Allow mixture to settle for 5 minutes – answer questions while you wait.
8. DNA will rise into the alcohol layer from the pea layer. You can use a wooden stick
to draw the DNA into the alcohol. Alcohol is less dense than water, so it floats on
the top. The protein and grease parts that we broke up with detergent and enzymes
fall to the bottom in the watery layer, while the DNA stays at the top in the alcohol
layer.
I don’t think I’m seeing DNA. What should I be looking for?
Look closely. Your DNA may be between the two layers of alcohol and soap. Try to help
the DNA rise to the top, alcohol layer. Dip a wooden stick into the soap solution and
slowly pull the white strings/clumps into the alcohol layer. Also, look very closely at
the alcohol layer for tiny bubbles. Even if your yield of DNA is low, clumps of DNA may
be loosely attached to the bubbles.
Lab based on “How to extract DNA from Anything Living!” http://learn.genetics.utah.edu/units/activities/extraction/
Name: Date: Period:
DNA Extraction Lab
Student Worksheet
Name:____________________________ Period:______ Date:______
Results/Analysis:
1. Were you able to see your DNA? Yes / No
2. Give 2 reasons why some people may have more DNA visible than other people?
• _____________________________________________________________
• _____________________________________________________________
3. Draw and color the test tube with the DNA in it. Label the DNA, soap layer and alcohol
layer.
Why add salt? What is its purpose?
Salty water helps the DNA precipitate (solidify and appear) when alcohol is added.
Why add soap?
The soap breaks down the fats in the cell membranes and nuclear membranes. This
releases the chromosomes containing DNA from the nucleus. As result, the DNA is
released.
Why does the DNA clump together?
DNA precipitates when in the presence of alcohol, which means it doesn’t dissolve in
alcohol. This causes the DNA to clump together when there is a lot of it. And, usually,
cells contain a lot of it! For example, each cell in the human body contains 46
chromosomes (or 46 DNA molecules). If you lined up those DNA molecules end to end,
a single cell would contain six feet of DNA! If the human body is made of about 100
trillion cells, each of which contains six feet of DNA, our bodies contain more than a
billion miles of DNA!
How do we know that the white stringy stuff is DNA?
There is a protocol that would allow you to stain nucleic acids (DNA), but the chemical
used would need to be handled by a teacher or an adult. So, for now, you’ll just have
to trust that the molecules precipitating in the alcohol are nucleic acids.
Can I use a microscope to see the DNA that I extract?
Unfortunately, a microscope will not allow you to see the structure of the DNA
molecule. You’ll only see a massive mess of many, many DNA molecules clumped
together. In fact, the width of the DNA double helix is approximately one billionth of a
meter! This is much too small to see, even with the most powerful microscope.
Instead, a technique called X-ray crystallography can be used to produce a picture of
the DNA molecule. It was by looking at such a picture (taken by Rosalind Franklin) that
James Watson and Francis Crick were able to figure out what the DNA molecule looks
Like.
Questions
Name
Level 1
1. The purpose of adding salt to the peas was to:
__________________________________________________________________
Per
Date
2. The soap solution was used to ____________________________________________
3. The DNA is found in the __________ of cells.
4. In humans, the DNA is packaged into ___ chromosomes.
5. True or False
Pea cells contain the entire genome for the plant — all the genetic information
necessary to build a new pea plant.
6. True or False
DNA extraction can be a useful tool for scientist studying genetics.
7. Why does DNA clump together?
__________________________________________________________________
8. Why can’t we use a microscope to view DNA?
__________________________________________________________________
9. If you lined up DNA that is present in all cells of our bodies end to end, how long would this
molecule be? _____________________________________________________
Level 2
1. What did the detergent do to the cells containing the DNA?
2. Why must we add the enzymes to the mixture in order to extract the DNA?
3. Name three other types of cells one might extract DNA from?
Level 3
1. List 3 ways in which DNA extraction can be useful and why.
Level 4
1. How could the procedure be different to extract the DNA from animal cells and prokaryotic cells?
Level 5
Competencies
S01. Scientific
Investigation:
Complete projects that
demonstrate
understanding of
different kinds of
scientific investigation,
controlled experiment,
fieldwork,
experimental design,
and secondary
research.
Emerging
(Remember)
Absence
of
Evidence
0
1
Define different types
of a scientific
investigation. Recite
or label the
characteristics and/or
steps of a scientific
investigation.List
observable data.
Capable
(Understand/Apply)
Explain how different types of
investigations can be used to
answer scientific questions
and investigations.Develop a
hypothesis, conduct a predesigned scientific
investigation, and describe the
results.
Explain how different types of
investigations can be used to
answer scientific questions
and investigations.
Collect and classify observable
data.
Proficient
(Analyze)
Advanced
(Evaluate)
Mastery
(Create)
3
Conduct a scientific
investigation and
analyze the results to
determine if it
satisfactorily addresses
the hypothesis.
4
Evaluate the design
and results of an
investigation to
determine if it
satisfactorily
addresses the
hypothesis.
5
Develop and
conduct an
investigation to test
a hypothesis and
analyze the
results.
Advanced
(Evaluate)
4
Evaluate the
strengths and
limitations of various
scientific tools and
techniques and how
they may impact the
outcomes of
scientific
investigations.
Mastery
(Create)
5
Design and
conduct an
experiment that
uses appropriate
tools and technique
to gather, analyze
and interpret
relevant data.
1. Create and perform a lab extracting DNA from a different type of cell.
Rubric for Grading
Competencies
Absence
0
S02. Scientific
Tools: Demonstrate
appropriate use of
tools and techniques
to gather, analyze
and, interpret data.
Capable
(Understand/Apply)
2
The student makes some
significant errors when
performing the skill or process
important to the topic but still
accomplishes a rough
approximation of the skill or
process.
The student makes so many
errors in performing the skill or
process important to the topic
that he or she cannot actually
perform the skill or process.
B.1.3
Competencies
P01. Selfmanagement:
Take responsibility for
changing personal
behaviors or acquiring
skills that lead to both
social and academic
success.
Capable
(Understand/Apply)
2
Explain how different tools can
be used to answer different
scientific questions.
Use tools and techniques to
measure, gather, and record
scientific data.
Emerging
(Remember)
1
B.1.2
Emerging
(Remember)
1
List scientific tools
and techniques and
describe how to use
them.
B.1.4
Proficient
(Analyze)
3
Conduct an experiment
using appropriate tools
and techniques, record
results, and analyze
margin of errors in
measurements.
Indiana State Standards Rubric
Proficient
(Analyze)
3
The student can perform the skill
or process important to the topic
without making significant errors.
Advanced
(Evaluate)
4
The student can perform the skill
or process important to the topic
with no significant errors and with
fluency. Additionally, the student
understands the key features of
the skill process.
Mastery
(Create)
5
The student can perform the skill
or process important to the topic
with no significant errors and with
fluency. Additionally, the student
has a detailed understanding of
the process and is able to critique
and/or defend the use of the
process in a variety of situations.
B.1.12
Abse
nce
Emerging
(Remember)
0
1
Define responsibilities for
changing personal
behaviors or acquiring
skills that lead to both
social and academic
success (e.g. turning
work in on time, arriving
to class on time).
Capable
(Understand/Apply)
2
Understand and apply
responsibilities for changing
personal behaviors or
acquiring skills that lead to
both social and academic
success.
Proficient
(Analyze)
3
Connect responsibilities
for changing personal
behaviors or acquiring
skills that lead to both
social and academic
success.
Advanced
(Evaluate)
4
Assess
responsibilities for
changing personal
behaviors or
acquiring skills that
lead to both social
and academic
success.
Mastery
(Create)
5
Modify and
combine
responsibilities for
changing personal
behaviors or
acquiring skills that
lead to both social
and academic
success.
DNA Background
Since DNA is an essential molecule to all living things (with the exception of some viruses), it is not
surprising that elaborate mechanisms have evolved to protect it. To extract DNA successfully, it is
helpful to understand these protective mechanisms.
The simplest organisms, prokaryotes, which include bacteria, do not have the protection of a
membrane-bound nucleus. Rather, the DNA clings to an in-folding of the inner cell membrane and is
protected from invading viral DNA by restriction enzymes that cut foreign DNA into small pieces.
Methyl groups that are attached to its DNA protect the host cell from its own defenses. Methyl
groups prevent restriction enzymes from cutting DNA.
As organisms become more complex, so do the mechanisms that protect their DNA. Eukaryotic DNA
is contained within a membrane-bound nucleus. Plants have additional protection from a cell wall. All
eukaryotes have DNAse enzymes in the cytoplasm that cut DNA. To produce spoolable DNA in the
laboratory, it is necessary to denature the DNAses before interrupting the nuclear membrane, often
by using heat or pH changes. DNA is a relatively sturdy molecule but its tremendous length makes it
prone to breaking once it is away from its protective environment. If the DNA is broken or sheared in
too many places, it won’t spool. It is important to be gentle in the last steps of DNA extraction and to
avoid violent shaking or vortexing that will shear the DNA.
Historical Milestones
1869
Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the nuclei of
human white blood cells. This substance will later be called deoxyribonucleic acid, or DNA.
1912
Physicist Sir William Henry Bragg and his son, Sir William Lawrence Bragg, discover that they can
deduce the atomic structure of crystals from their X-ray diffraction patterns. This scientific tool will be
key in helping Watson and Crick determine DNA's structure.
1924
Microscope studies using stains for DNA and protein show that both substances are present in
chromosomes.
1928
Franklin Griffith, a British medical officer, discovers that genetic information can be transferred from
heat-killed bacterial cells to live ones. This phenomenon, called transformation, provides the first
evidence that the genetic material is a heat-stable chemical.
1944 Oswald Avery, and his colleagues Maclyn McCarty and Colin MacLeod, identify Griffith's
transforming agent as DNA. However, their discovery is greeted with skepticism, in part because
many scientists still believe that DNA is too simple a molecule to be the genetic material.
1949
Erwin Chargaff, a biochemist, reports that DNA composition is species specific; that is, the amount of
DNA and its nitrogenous bases varies from one species to another. In addition, Chargaff finds that
the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount
of cytosine in DNA from every species.
1953
James Watson and Francis Crick discover the molecular structure of DNA.
1962
Francis Crick, James Watson, and Maurice Wilkins receive the Nobel Prize for determining the
molecular structure of DNA.
(Historic milestones link: Access Excellence,
www.gene.com/gene/research/biotechnology/significant-milestones.jsp)
Some basic, but cool, chemistry…
DNA is the largest known molecule. A single unbroken strand can contain millions of atoms. When
DNA is released from a cell it typically breaks up into tiny strand fragments. These tiny fragments
have a slightly negative electric charge. Salt ions, common in many solutions, are attracted to the
negative charges on the DNA fragments and prevent them from adhering to one another. By
controlling the salt concentration of the solution containing the DNA fragments, DNA can remain
fragmented or become very “sticky” and form large globs of molecular material.
Releasing the DNA…
The first step in obtaining DNA from any organism, be it a plant, animal, fungi, archae or bacterium,
is to release the DNA from a cell. Detergents and soaps break down cell membranes, releasing the
DNA, and they also break up proteins that may harm the DNA. Protein enzymes, or proteases, like
those in contact lens solution or in “Ultra” forms of laundry detergent, can be used to further destroy
proteins.
DNA on a stick…
Once the DNA fragments are released into solution, the DNA can be spooled together by using icecold isopropyl alcohol. Alcohol allows DNA fragments to stick together, or precipitate, producing a
blob of DNA that you can examine. When a small layer of alcohol is added to the top of a solution
containing cellular fragments and DNA, it will form an interface where the DNA will precipitate,
allowing it to be captured, or spooled, onto a wooden stick or glass rod. Although this method is
effective, the DNA produced is by no means pure; other materials such as protein and cell fragments
are carried along.
DNA Genetic material found in all of our cells. DNA is an abbreviation for deoxyribo nucleic acid.
Lysing The process of breaking open cells.
Negative Control A sample with no DNA (e.g. water).
Positive Control A known source of DNA.
Precipitation The process of bringing compounds out of solution. DNA comes out of solution in
alcohol, so visible DNA forms at the surface where the alcohol and cell sample meet.
Restriction Enzymes Enzymes that cut DNA at specific base sequences.
The University of Maryland Biotechnology Institute’s SciTech Education Program in partnership with Maryland Sea Grant
Extension Program