Download In search of a cancer gene

In search of a cancer gene
In search of a cancer gene
 You will search for a gene by
 Pedigree Analysis
 Gel electrophoresis
 Sequencing Data
 Pedigree of “Valerie’s” family (3-130)
 Label Photo with typed figure legend and data
interpretation
 ID “children” that carry mutation from DNA sequences
Hereditary versus Somatic
Mutations
Germline
Somatic
Some well known germline
mutations that result in cancer
are:
Li-Fraumeni Syndrome
What does the pedigree of a LiFraumeni patient look like?
 A sarcoma patient is included
 At least two immediate relative with other cancers before
the age 45
 As well as multiple cancers in other family members
 Your lab manual provides a typical LF pedigree
Which gene is involved in the
Li-Fraumeni syndrome?
 The p53 gene
 F.P. Li, J.F. Freemeni, Jr.:
Soft-tissue sarcomas, breast cancer and other
neoplasm's: A familial syndrome?
Annals of Internal Medicine, Philadelphia,
October 1969, 71 (4): 747-752.
What are some characteristics
of p53?
 “p” arm of chromosome 17
 nuclear phosphoprotein
 transcription factor
 tumor suppressor gene
 “hot spots”
– Exons 5-8 are hot spots
P53 Mutations
Point Mutations
Mutation alters digest pattern
Identifying Li-Fraumeni in
three ways:
 Family History: leads to a pedigree
 Germline mutations: restriction digests, followed by gel
electrophoresis
 Hot Spots: sequencing
Let’s look at these steps
individually…
The first step will be assign a pedigree
to family containing patient “Valerie”
Use the appropriate pedigree symbols as
Outlined on 3-120.
 For example:
Female free of cancer
Male free of cancer
The second step is to identify
patterns of the restriction digest
for patient Valerie.
Step 2: Restriction Digest
Analysis of Patient: “Valerie”
 Ladder
 Wild Type DNA (gold standard)
 Patient Peripheral Blood WBC DNA
 Patient Tumor DNA
 Patient Non-Tumor Breast Tissue
Step 3: Identify the “hot spot” in
Valerie’s children.
Was this mutation inherited by her
children?
Read the sequences of the 5 films
–
–
–
–
–
Film 1 Justin age 16
Film 2 Sheila age 14
Film 3 Robert age 10
Film 4 Angela age 8
Film 5 Anthony age 6
Summary: Today you will…
perform gel electrophoresis
Read sequencing films
Practice a pedigree for Valerie’s family
Points on Gel Electrophoresis
 4-5 groups
 Each group should pick up 5 samples labeled A,
B, C, D and E
 Load 20 ul
–
–
–
–
–
A: Ladder
B: wild type DNA “gold standard” (non-patient DNA)
C: Patient Peripheral Blood DNA
D Patient Tumor DNA
E Patient Non-tumor Breast Tissue
Gel Electrophoresis
 Heat samples 2 min. at 65 C in water bath in
floating racks
 Add 20 ul per well
 Run for 75-125 volts, 20-25 minutes
 Photograph results
What you hand in:
 Photograph with a legend and very brief
explanation of the result
 Family pedigree
 Sequence of children and identify:
– Who has a mutation
– Where the mutation is (simply highlight with marker)