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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.

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In search of a cancer gene

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  1. In search of a cancer gene

  2. 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 In search of a cancer gene

  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): 747-752.

  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 3-120. • 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” inValerie’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 75-125 volts, 20-25 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)

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