Cancer Investigators: Medical Diagnostics in Your Classroom

Transcription

1 Cancer Investigators: Medical Diagnostics in Your Classroom Thomas Cynkar Paul Miller Edvotek, Inc.

2 EDVOTEK Biotech The Biotechnology Education Company Celebrating 30 years of science education! EDVOTEK Educatio

3 EDVOTEK The Biotechnology Education Company Experiments Reagents Equipment Resources

4 Cancer Impacts Each & Every One of Us Personally, relative, or friend. By studying the different aspects of this disease, we can help students gain a better scientific understanding of cancer. In addition, the topic of cancer can serve as a relatable and accessible starting point to discuss many aspects of cell development, the cell cycle, molecular biology, biochemistry, genetics Inspire a young person who could maybe someday research & find a cure.

5 Cancer is Responsible for Almost 25% of All Deaths in the USA Breast cancer Colon cancer Ovarian cancer Cervical cancer Prostate cancer Blood cancers Brain cancer Pancreatic cancer Lung cancer Liver cancer Skin cancer Bladder cancer Bone cancer Thyroid cancer

6 Cancer as a Result of Uncontrolled Cell Division

7 Today s Agenda: 1. Electrophoresis analysis of DNA from a patient suspected of carrying mutations in her p53 tumor suppressor gene. Results will indicate the likelihood of her developing cancer. 2. Compare normal vs. cancer cells on microscope slides 3. Review a family Pedigree 4. Look at DNA Sequencing data corresponding to the family pedigree.

8 Overview of Agarose Gel Electrophoresis

9 What equipment do I need?

10 What s in my Read-to-Load electrophoresis kit?

11 Let s run our gels! Load 25 microliters of sample per well. Lane Sample 1 EdvoQuick DNA Ladder 2 Control DNA 3 Patient Peripheral Blood DNA 4 Patient Breast Tumor DNA 5 Patient Normal Breast Tissue DNA

12 Electrophoresis Chambers for Classrooms of all Sizes Cat. # 502/504 Model M12 two 7x7cm gels + one 7x14cm gels Cat. # 515 Model M36 Six gels

13 Power Supplies Provide Current for Electrophoresis Cat. #509 DuoSource 150 (75/150 V) Cat. #5010 QuadraSourceTM (10-300V)

14 Overview of Agarose Gel Electrophoresis

15 Analyzing a Family Pedigree

16 Analyzing Valarie s Family Pedigree CN, 3 OS, 18 Her mother, Diane, was diagnosed and treated for breast cancer at the age of 39. Valerie did not know that Diane had a sister, Mabel, who died at age 2. Diane's brother, James (age 40), underwent surgery and chemotherapy for colon cancer. Her maternal grandmother, Elsie, died at age 42 from bilateral breast cancer. Her maternal grandfather, Elmer, was free of cancer and is 88 years old. Her maternal cousin, Patrick (son of James), died of brain cancer at 14. Her cousin (Patrick's sister), Jane, was diagnosed with childhood leukemia and subsequently died at age 2. Patrick's two other brothers, Richard (age 28) and Curtis (age 30), are in good health and free of cancer. Valerie's sister Nancy (age 38) is cancer free.

17 Changes in Cell Morphology

18 Changes in Cell Morphology

19 Changes in Cell Morphology Staining pre-fixed cells using Methylene blue and Eosin Edvo-kit #990

20 Changes in Cell Morphology

21 Analyzing DNA Sequencing Results

22 Electrophoresis Separates DNA Fragments By Size The sugar-phosphate backbone of DNA has a strong negative charge. A B C When an electrical current is passed through the gel, the current drives the DNA fragments through the gel towards the positive electrode.

23 Electrophoresis Separates DNA Fragments By Size The gel contains small channels through which the DNA can pass. A B C Small DNA fragments move through these holes easily, but large DNA fragments have a more difficult time squeezing through the tunnels.

24 Electrophoresis Separates DNA Fragments By Size Because molecules of different sizes travel at different speeds, discrete bands are formed. A B C After the current is stopped, the bands can be visualized using a stain that sticks to DNA. UV-reactive dyes simulate DNA fragments, eliminating poststaining time.

25 SybrSafe DNA Stain In-gel Staining Melt agarose and cool to 60 C. Add concentrated Sybr Safe stain to the molten gel at 1:10,000 dilution (5 µl per 50 ml agarose solution). Run DNA samples through gel no post staining or destaining necessary! Post-electrophoresis Staining Dilute concentrated stain to 1:20,000 (5 µl per 100 ml distilled water). After electrophoresis, place gel in tray. Cover gel with diluted Sybr Safe stain. Stain gel for minutes. Transilluminator #558 SybrSafe Stain #608

26 TruBlu Bluelight Transilluminator Optimized for SYBR Safe stained gels Large viewing area No harmful UV

27 Using the PCR-RFLP Method to Characterize p53 A B A B Cat. # 109 This region of the p53 gene can be amplified by PCR and characterized using Restriction Fragment Length Polymorphism (RFLP) analysis. The SNP in the gene adds a restriction enzyme cut site to the DNA.

28 Gel Results Normal p53 genes will not have a restriction enzyme cut site, and will run as a 4282bp band. A mutation will introduce the restriction site, resulting in digest DNA and the two shorter bands.

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