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DNA Fingerprinting & Genetic Counseling Spring 2017
WEEK 1 PREPARATION
1. Read about ‘restriction enzymes’ (RE’s) on page 234 of your textbook and on pages 56 – 57 of
the lab manual (“Introduction to DNA Fingerprinting”). Answer the following questions:
a.
b.
c.
d.
e.
What type of molecule are RE’s?
What do RE’s do to DNA?
What is a recognition site?
Where do RE’s come from?
How do they benefit this organism?
2. Preview “Separation of DNA by agarose gel electrophoresis” on page 75 of the lab manual, then
go to the DNA interactive web site ( http://www.dnai.org/index.htm ) and select “Manipulation”
then “Techniques” then “Sorting and Sequencing” (2nd row, top of the page), then “Gel
electrophoresis”. Read the descriptions as you click through the animation. Follow the
instructions (e.g., you need to click on the power supply to turn on the gel). You will be
simulating this technique in this lab and performing this technique later in the semester. (If the
link above is not working, a second website you can use to answer the questions below is:
http://learn.genetics.utah.edu/content/labs/gel/) Answer the following questions:
a.
b.
c.
d.
e.
What is the purpose of GE?
Gels are porous – why is this important?
Why do DNA fragments move through the gel?
What determines how fast or far a DNA fragment will move through a gel?
Why is blue dye added to the DNA sample?
3. Preview WebCutter on pages 58 – 62 of the lab manual. You will be doing this exercise in lab.
Read page 71 of the lab manual, “A day in the life of a genetic counselor”.
4. Read about gene mutations – small changes in the nucleotide sequence within one gene,
textbook section 10.16.
5. Use the University of Utah’s Learn.Genetics website
http://learn.genetics.utah.edu/content/disorders/ and the National Human Genome Research
Institute website http://www.genome.gov/19016930 to learn about genetic disorders. Answer
the following questions:
a. What are genetic disorders?
b. Specifically, what are single gene disorders?
c. What is a genetic counselor?
6. Visit the following site to familiarize yourself with how family pedigrees are constructed.
• http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/mendel/mendel9.htm
• You may wish to draw a pedigree of your own family (or a fictitious family) for practice,
starting with your grandparents (both pairs) and showing all of their children and
grandchildren.
DNA Fingerprinting & Genetic Counseling Spring 2017
7. There will be a prep quiz at the beginning of class. Review your notes!
8. Be sure to bring your lab manual and a laptop (if possible) with you to lab.
WEEK 1 IN LAB ACTIVITIES
•
•
•
Webcutter exercise, lab manual pages 58-62
Pedigree – analyze the family history of a couple concerned about the disease you are
researching; construct a pedigree based on the reported phenotypes for the couple’s entire
family, starting with the grandparents
If possible, determine the inheritance pattern (autosomal dominant, autosomal recessive or Xlinked recessive) of the disorder you are researching by reviewing the pedigree you constructed.
Add genotypes (wherever possible) to your pedigree – you will not be able to determine the
genotype of some individuals until you analyze the couple’s DNA next week.
WEEK 2 PREPARATION
1. Read over the following sections in Chapter 9 in your text: 9.2, 9.3, 9.8, 9.20, 9.21.
2. Preview Agarose Gel Electrophoresis on pages 74-76 of the lab manual, you will be doing this
activity in lab.
3. Each team member should be able to answer the following questions about the specific genetic
disease that you have been assigned (even if a particular question does not correspond to your
part of the presentation.) Bring this information back to lab! Suggested sources:
http://ghr.nlm.nih.gov/ (type disorder name into search bar)
http://learn.genetics.utah.edu/content/disorders/singlegene/
https://www.genome.gov/10001204/specific-genetic-disorders/
http://ghr.nlm.nih.gov/handbook/howgeneswork/genelocation (how geneticists indicate the
location of a gene*)
(Keep track of additional sources since you will need this information in your final presentation.)
a.
b.
c.
d.
e.
f.
What is the major symptom of this disease? What other symptoms are seen?
What organ(s) or system(s) of the body are most affected?
What is the typical age of onset?
What is the prognosis? (On average, about how long does a patient live?)
What is the name of the defective gene?
On which chromosome is the defective gene located? What is the specific location of
the defective gene*?
g. What is the name of the protein affected by this disorder, and what is its normal
function?
h. Is the disease-causing allele dominant or recessive? Autosomal or X-linked?
i. What type(s) of mutation are most commonly associated with disease (e.g., singlebase or multiple-base change; substitution, deletion, or insertion; etc.)?
j. How does the change in the protein caused by the mutation (i) lead to the major
symptom (a)?
DNA Fingerprinting & Genetic Counseling Spring 2017
k. What percentage of the population is affected with this disease? What percentage
are unaffected carriers?
4. There will be another prep quiz at the beginning of class. Review your notes!
WEEK 2 IN LAB ACTIVITIES
•
•
•
•
•
Determine who is responsible for each part of your presentation that is due next week
Complete a pedigree analysis activity
On Blackboard during week 2 you will have allele maps posted for each disease. Use the allele
maps (see analysis of restriction fragments starting on page 76) to predict the bands you would
expect to see if an individual is homozygous normal, heterozygous or homozygous diseased for
your disorder. We will call this drawing a “mock gel”.
Load the RE digested DNA samples from your family members on the gel as instructed. After
the gel runs, use the “mock gel” to interpret the bands on the gel. Determine the genotype and
phenotype of each individual.
Complete the pedigree with genotypes of the remaining family members (use a ? for alleles that
cannot be determined with certainty, for example “F?” )
WEEK 3
1. Poster/PowerPoint presentations and oral reports; final assessment in lab
12.2 Enzymes are used to "cut and paste” DNA
VISUALIZING
THE CONCEPT
To understand how DNA is manipulated in the
laboratory, you need to learn how enzymes cut and
paste DNA. The cutting tools are bacterial enzymes called
restriction enzymes. Biologists have identified hundreds of
different restriction enzymes, each of which recognizes a particular
short DNA sequence, which is called a restriction site. After a
restriction enzyme binds to its restriction site, it cuts both strands
of the DNA at precise points within the sequence-like a pair of
highly specific molecular scissors--yielding pieces of DNA called
restriction fragments. All copies of a particular DNA molecule
always yield the same set of DNA fragments when exposed to the
same restriction enzyme. Once cut, fragments of DNA can be
pasted together by the enzyme DNA ligase. The techniques
outlined here form the basis of many genetic engineering
procedures that involve combining DNA from different sources.
A restriction site is usually
4-8 nucleotide pairs long.
Restriction site
ma
DNA
入
GAATTC
CTTAAG
Restriction
enzyme
The restriction enzyme shown here, called
EcoRI, is found naturally in E. coli bacteria.
EcoRI recognizes the DNA sequence
GAATTC and always cuts it the same
way-between the bases A and
G-producing restriction fragments.
G
CTTAA
AATTC
G
In bacteria, restriction enzymes play
a defensive role, chopping up foreign
DNA, the cell's own DNA is protected
by the addition of methyl groups.
Sticky end
Gene of
interest
A piece of DNA from another source (the
gene of interest) is cut by the same
restriction enzyme and added to the first
DNA. Both molecules of DNA are cut
unevenly, yielding "sticky ends," single-
stranded extensions from the double-
stranded fragments.
AATTC
G
G
The sticky ends from the two different
DNA molecules are complementary to
one another because they were cut by
the same enzyme.
СТТАА
Sticky end
The complementary ends on
the two different fragments
stick together by base pairing.
GAATTC
CITAAG
GAATTC
CITAAG
Sticky ends are the key to joining
restriction fragments from different sources:
Hydrogen bonds (not shown) form base pairs
that hold the two strands together.
DNA ligase
The temporary union between the DNA
fragments is made permanent by DNA
ligase, which creates new covalent
bonds that join the sugar-phosphate
backbones of the DNA strands.
Recombinant DNA
?
What are "sticky ends"?
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234
CHAPTER 12
DNA Technology and Genomics
Unit #4: A day in the life of a genetic counselor
WebCutter Exercise
Unit #4: A day in the life of a genetic counselor
DNA
In this unit, you will
Learn about some of the basic methods used to manipulate and analyze DNA
Carry out other activities selected by your instructor to solidify your knowledge of
restriction enzymes and its application in DNA fingerprinting
INTRODUCTION TO DNA FINGERPRINTING
Restriction enzyme digestion of DNA
Every organism carries within it one or more nucleic acid polymers that contain a coded
message with instructions on how to make more of the same organism. These nucleic acid
polymers are the organism's genome, which contains all the genes an organism needs to
metabolize, grow, and reproduce. In all organisms (except for some viruses), this genomic
material is DNA. To study the individual genes of any organism it would be useful to purify
its genome and to isolate the individual genes therein. In the last 30 years, techniques for
cutting out individual genes from genomic DNA, pasting them together in new
combinations, and amplifying them inside growing cells have made the molecular biology
revolution possible. The crucial reagents in this process are restriction enzymes (RE),
which cut DNA at specific sequences (called "RE recognition sites") located randomly
along DNA strands. Because of this randomness, a cell's genome may contain none to
thousands of cut sites for any given restriction enzyme. Thus, when a linear strand of
DNA is treated ("digested") with a particular restriction enzyme, the number of
fragments generated equals the number of unique cut sites plus one. Because of the
random placement of RE recognition sites, digestion of a particular strand of DNA with a
particular restriction enzyme will produce many fragments of varying sizes, resulting in a
characteristic pattern of fragments for that enzyme. This pattern is called a restriction
map.
TI
CT G C AG
G|| А А т т C
C C
G G
-030
030
C T T A AG
G|A C GT C
G GG
1
|
|
ECORI
PstI
Smal
Figure 1. Restriction enzyme (RE) recognition sites. The recognition site (formed by a specific
sequence of six nucleotides) for three different restriction enzymes is shown. Depending on the
enzyme, the cut can occur in one of three ways, as illustrated here.
Unit #4: A day in the life of a genetic counselor
WebCutter Exercise
Of the three billion nucleotide base pairs that make up the human genome, only 0.1%,
(about three million base pairs) differs from one person to the next. Differences in the
genome that are commonly seen in the population are called DNA polymorphisms (poly =
many: morph = form). Additionally, differences in the number and/or position of RE sites
in a given gene are often seen between the gene's normal form and the disease-causing
mutated form. Individuals may have just normal gene copies or may have one or more of
the mutated gene copies in their DNA.
If a polymorphism or disease-causing mutation causes a change in an RE recognition site,
then RE digestion of DNA from an individual with one version of that polymorphism or gene
will produce a set of fragments that differ in number and size from the fragments
obtained when DNA from someone with a different version of the polymorphism or gene is
digested with the same RE. Thus, it is possible to distinguish between individuals based on
their restriction fragment length polymorphisms, or RFLPs. Therefore, the pattern of
fragments produced by digesting an individual's genomic DNA with a particular enzyme can
be used to identify that individual, for example in forensics. Genomic DNA samples from
two different individuals will differ in the restriction fragments produced, enabling tissue
samples from the two individuals to be distinguished from one another, just as individuals
can be distinguished by the patterns of ridges on their fingertips. Likewise, differences
in the pattern of fragments from the DNA of a particular gene can be used for diagnosis
of illness.
Separation and Analysis of DNA fragments produced by Restriction Enzyme
Digestion - see Unit 4: "A Day in the Life of a Genetic Counselor"
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