BIOL 207 Biology of Cancer
DNA Testing Workshop
Fall 2005
Today you will see demonstrations of an RFLP DNA test, automated DNA
sequencing, and fluorescence microscopy, set up as demonstrations in Room 4,
Science I. This room is the Biology Research Lab; Dr. Bachman and other
faculty and students carry out cell/molecular biology and genomics research
using this equipment. Many of these methods are also important for cancer
research and diagnostics.
The assignment will involve analyzing the pedigree of a family with an
inherited risk for cancer, interpreting a DNA test for cancer in one of the family
members, and analyzing a DNA sequencing result for a mutation associated with
cancer.
Workshop Stations. Room 4, Science I
I. DNA Testing: Hereditary Li-Fraumeni Syndrome
Simple screening for common mutant forms of tumor suppressor genes associated with
cancer can be done by analysis of restriction fragment length polymorphisms (DNA
fragments of different lengths separated by agarose gel electrophoresis).
1. Demonstration of micropipets and practice loading an agarose gel to separate
DNA fragments. Lanes of the gel:
Lane
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2
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Contents
Standard DNA fragments
Control DNA (normal individual)
Patient Peripheral Blood DNA
Patient Breast Tumor DNA
Patient Breast Normal DNA
Purpose
size markers
to show the normal pattern
to show the inherited p53 status
to show whether tumor has mutant p53
for comparison with the tumor DNA
2. Electrophoresis of the gel.
3. Visualization of DNA bands: Ethidium Bromide stained gel.
II. DNA Sequencing
Highly specific tests for variants in the sequence of tumor suppressor genes are
available for several hereditary cancers. These typically use the same DNA sequencing
chemistry used for the human genome project. In the dideoxy sequencing method, DNA
chains of different lengths are produced from the gene of interest and the order of bases
can be determined after separating them by denaturing gel electrophoresis.
1. Demonstration of automated DNA sequencing system (capillary system).
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2. Analysis of DNA sequencing traces.
3. Cancer databases.
III. Fluorescence microscopy
1. Demonstration of fluorescence microscope/digital imaging system:
Dr. Bachman's research with human cervical cancer cells.
2. Microscopic analysis of solid tumors and leukemias.
3. Cancer cytogenetics/chromosome painting.
Assignment: DNA Testing (20 pts.)
Please briefly type or neatly print your answers to the following questions
on separate pages and hand in by Friday Dec. 2, 2005. Questions are based on
the DNA workshop, the heredity handout and reserve readings, but feel free to
consult any other sources you wish, as long as you cite them.
1. Construct a pedigree of the family afflicted with cancer based on the
information on the attached pages. Be sure to use conventional genetic symbols
to symbolize marriages, individuals affected by the trait (cancer), males, females,
children, unknown individuals, etc. Using the symbol T for the normal tumor
suppressor gene and t for the mutant gene, and recalling that each individual
has two copies of the gene, indicate each individual's genotype for p53.
2. Describe the pattern of DNA bands seen in non-cancerous tissues for an
individual with heredity Li-Fraumeni syndrome (patient Valerie), who has a defect
in the p53 tumor suppressor gene. What is the likelihood of her transmitting the
defective gene to each of her children?
3. p53 is among the most important of the tumor suppressor genes identified so
far.
a. Which cancers have the highest incidence of p53 mutation associated with
them?
b. Give at least two critical functions for normal p53 in the cell.
c. Which regions of the p53 gene are the most likely to be mutated in human
cancers?
d. How does this information help us to design treatments for cancers in which
p53 plays a critical role?
4. Answer the following questions for one of the following tumor suppressor
genes
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BRCA1
BRCA2
APC
Rb
a. What risk factors would a patient that goes in for DNA testing for this gene
typically have?
b. What are the more common types of mutations in the gene (point mutations,
insertions, deletions and location)?
c. What advice should be offered for a positive DNA test?
5. The following panels show regions of the DNA sequence surrounding a single
nucleotide polymorphism identified in the DNA sequence of a tumor suppressor
gene as compared to the normal gene. Assuming the reading frame of the
mRNA produced is in the same register as the left side of the sequence panel,
the control produces an mRNA with the sequence:
5' AGG CCC UUG AAA
what is the affect of the mutation on the gene in the cancer patient?
6. The following result is from fluorescence in situ hybridization (FISH) of an
individual with chronic myeloid leukemia (CML). Hybridization with two probes is
shown, bcr (green) and abl (orange).
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a. What does the pattern indicate about the association of the bcr and abl
genes?
b. What would be the prognosis for an individual with this diagnostic pattern?
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