Insearchofacancergene

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,In search of a cancer gene,1,In search of a cancer gene,You will search for a gene by,Pedigree Analysis,Gel electrophoresis,Sequencing Data,Pedigree of “Valeries” family (3-130),Label Photo with typed figure legend and data interpretation,ID “children” that carry mutation from DNA sequences,2,Hereditary versus Somatic Mutations,Germline,Somatic,3,Some well known germline mutations that result in cancer are:,Li-Fraumeni Syndrome,4,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,5,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 neoplasms: A familial syndrome?,Annals of Internal Medicine, Philadelphia, October 1969, 71 (4): 747-752.,6,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,7,P53 Mutations,Point Mutations,Mutation alters digest pattern,8,Identifying Li-Fraumeni in three ways:,Family History: leads to a pedigree,Germline mutations: restriction digests, followed by gel electrophoresis,Hot Spots: sequencing,9,Lets look at these steps individually,10,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,11,The second step is to identify,patterns,of the restriction digest for patient Valerie.,12,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,13,Step 3: Identify the “hot spot” inValeries 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 6,14,Summary: Today you will,perform gel electrophoresis,Read sequencing films,Practice a pedigree for Valeries family,15,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,16,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,17,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),18,19,