SAN DIEGO MESA COLLEGE
School of Natural Sciences & Mathematics
Intro Molecular & Cellular Biology Lab (BIO 210A); Instructor: Elmar Schmid, Ph.D.
R
RFFLLP
PA
Annaallyyssiiss
Objectives
_______________________
(Student Name)
After completion of this lab you should
1. understand the principle behind the restriction fragment length polymorphism
(RFLP) method and be able to describe the individual steps
2. understand the importance of a suitable DNA probe necessary to trace the
presence of a certain gene during RFLP analysis
3. predict from hypothetical human pedigree charts and based on the restriction
fragment pattern of a certain gene the genotypes for the individual family
members
1. Procedure: RFLP Analysis
1. Write down the individual steps of the RFLP method in the right sequence
(from the first to the last step) on the lines below.
1.
2.
3.
4.
5.
6.
__________________________________
__________________________________
__________________________________
__________________________________
__________________________________
__________________________________
2. At which step of the RFLP method do you need the single-stranded DNA
probe which recognizes (by hybridization) the gene you are examining?
Step ___
=
________________________________
3. Which step of the RFLP method separates the DNA fragments according to
length?
Step ___
=
________________________________
After completion of this method, the shorter DNA fragments will be found at
the _____________ of the _______________ matrix.
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SAN DIEGO MESA COLLEGE
School of Natural Sciences & Mathematics
Intro Molecular & Cellular Biology Lab (BIO 210A); Instructor: Elmar Schmid, Ph.D.
4. Look at the Figure below which shows the simplified restriction site map of a
certain human gene and of a mutated version thereof; the red arrows in the
Figure below depicts the known restriction sites for the restriction enzyme
Bgl-II; it further shows the hybridization site of a certain DNA probe (= purple
colored line) you want to use to do RFLP analysis with the collected DNA of
family members to determine their genotypes.
6,800 bp (= 100%)
DNA probe
Gene
Normal gene
(N- allele)
Gene
*
= Bgl-II restriction sites
*
= Point mutation
Mutant gene
(M- allele)
a. Write down the numbers of expected DNA fragments and their lengths for
the normal gene and for the mutated version of the gene.
Gene
No. of DNA fragments
Lengths (bp)
Normal
_____
__________
Mutated
_____
__________
Calculations:
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SAN DIEGO MESA COLLEGE
School of Natural Sciences & Mathematics
Intro Molecular & Cellular Biology Lab (BIO 210A); Instructor: Elmar Schmid, Ph.D.
b. Draw the expected DNA banding pattern you would observe after completion
of the RFLP method for a person with two normal genes (= homozygous
normal; NN), a person with one normal and one mutated gene (=
heterozygous; NM) and a human individual with two mutated genes (=
homozygous mutated; MM) into the gray box below.
(Label the blot by showing the genotypes and the DNA fragment sizes for
these three “model individuals”)
DNA
(bp)
Genotype
RFLP pattern
(after Southern Blotting)
NN
NM
MM
5. Carefully study the print-out of Lab Assignment Sheet II and answer the
following questions below.
a. What kind of inheritance pattern do we observe for the heritable disorder “M”
in that family (dominant/recessive/autosomal/X-linked/other? )
________________________ inheritance
b. Write down the genotypes for the depicted family members at the bottom of
the shown RFLP blot box.
(Use the lower case “m” letter for the mutated version of the MRE gene and
the capitalized “M” letter for the normal MRE gene!)
c. Use the information supplied by the MRE gene map (= shown restriction
sites and DNA probe hybridization site) on the left side of Lab Assignment
Sheet II to complete the Southern Blot (on the same page) by correctly
depicting the RFLP pattern for each family member under examination.
(Use little dashes to symbolize the DNA fragments)
Remember: Short DNA fragments are at the bottom of the Southern Blot!
d. Complete the Lab Assignment Sheet II by correctly labeling the MRE gene
fragments in the Southern Blot.
(Which DNA fragment indicates the mutated MRE gene version?)
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SAN DIEGO MESA COLLEGE
School of Natural Sciences & Mathematics
Intro Molecular & Cellular Biology Lab (BIO 210A); Instructor: Elmar Schmid, Ph.D.
6. Carefully study the print-out of Lab Assignment Sheet III which shows the
pedigree of a family which is affected by a heritable form of mental
retardation; complete the following assignments and answer the following
questions below.
a. What kind of inheritance pattern do we observe for this heritable trait “MR” in
that family (dominant/recessive/X-linked/other?)
________________________ inheritance
Why? (Argue your answer)
____________________________________________________________
____________________________________________________________
b. Complete the pedigree shown in the assignment sheet III by showing the
heterozygote family members and/or carriers of that family
c. Draw the genotypes for the indicated family members one would expect after
Southern Blotting using a chromosome-specific DNA probe (= AGTR probe)
into the empty grey box
- use black dashes to indicate the (longer) normal AGTR gene fragment and
red dashes to indicate the position of the shorter mutant AGTR fragment
- an example is shown in the Southern blot on the very right
d. What is the chance of family member III-7 to give birth to a mentally retarded
son if she marries a male with no family history for that disorder?
Probability p = ________________
7. The pedigree and RFLP analysis as shown in Lab Assignment Sheet IV was
prepared when the individuals II-1 (age 46) and III-6 (age 39) requested
medical advice about their risks to develop breast or ovarian cancers in their
lives and to get information about possible treatment options. As you can see
in the established pedigree, both, III-1 and III-6 had every right to be
concerned. III-1’s grandmother (= I-2) died of ovarian cancer at the age 59,
and individual III-6’s grand mother (= I-2), mother (= II-4) and her sister (= III-5)
died of either breast or ovarian cancer before reaching the age of 60.
a. What kind of inheritance pattern do we observe for the heritable trait “breast
or ovarian cancer” in that family (dominant/recessive/X-linked/other?)
________________________ inheritance
4
SAN DIEGO MESA COLLEGE
School of Natural Sciences & Mathematics
Intro Molecular & Cellular Biology Lab (BIO 210A); Instructor: Elmar Schmid, Ph.D.
b. What is the most likely genotype of individual III-1 based on the study of the
pedigree? Mark the genotypic pattern of her into the corresponding lane of
the shown RFLP blot in your lab assignment sheet #4 (dashed line)
- Glue: Look at the genotype and the age of her mother (= II-2) and use the
info from the Risk Assessment Chart below!
Genotype (III-1) = _______
c. What is the risk of individual III-1 (age 46) to develop breast or ovarian
cancer by the age 50?
(Use the Risk Assessment Chart below to give an answer)
Probability (III-1) = _____ %
d. Based on the finding after RFLP analysis, what is the genotype of individual
III-6? Does she have a mutated version of the BRCA1 gene in her genome?
Genotype (III-6) = ________ (= ________ zygous)
Presence of BRCA1 gene:
Yes _____
No _____
e. Based on the information supplied by the Risk Assessment Chart below,
what is individuals III-6 (age 39) to develop breast or ovarian cancer before
or at the age of 50?
Probability (III-6) = ______ %
f. Complete the Lab Assignment Sheet IV by writing down the genotypes of the
family members analyzed with the help of the RFLP method (empty box at
the bottom of the RFLP blot) and write down the missing information about
the BRCA1 gene
5
SAN DIEGO MESA COLLEGE
School of Natural Sciences & Mathematics
Intro Molecular & Cellular Biology Lab (BIO 210A); Instructor: Elmar Schmid, Ph.D.
BRCA1 Risk Assessment Chart
Genetic testing for BRCA1 mutation can help to assess the
risk for developing breast or ovarian cancer in women
C
a
n
c
e
r
P
r
o
b.
(p)
(%)
100
with BRCA1
mutation
50
with Family
history
without BRCA1
mutation
0
0
30
40
50
Age (years)
60
70
6