1. In search of a cancer gene

2. 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

3. Hereditary versus Somatic Mutations
Germline
Somatic

4. Some well known germline mutations that result in cancer are:
Li-Fraumeni Syndrome

5. What does the pedigree of a Li-Fraumeni 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

6. 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):

7. 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

8. P53 Mutations
Point Mutations
Mutation alters digest pattern

9. Identifying Li-Fraumeni in three ways:
Family History: leads to a pedigree
Germline mutations: restriction digests, followed by gel electrophoresis
Hot Spots: sequencing

10. Let’s look at these steps individually…

11. The first step will be assign a pedigree to family containing patient “Valerie”
Use the appropriate pedigree symbols as
Outlined on
For example:
Female free of cancer
Male free of cancer

12. The second step is to identify patterns of the restriction digest for patient Valerie.

13. 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

14. 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

15. Summary: Today you will…
perform gel electrophoresis
Read sequencing films
Practice a pedigree for Valerie’s family

16. 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

17. Gel Electrophoresis
Heat samples 2 min. at 65 C in water bath in floating racks
Add 20 ul per well
Run for volts, minutes
Photograph results

18. 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)