Mitochondrial Deletions Induced By UVB Irradiation Of Human Epithelial Cells IN VITRO

1. Mitochondrial Deletions Induced By UVB Irradiation Of Human Epithelial Cells IN VITRO By: Jing Jing He Mentor: Dr. Mark Steinberg The City College of New York

2. Introduction The Human Mitochondrial Genome • The 16 Kbp mitochondrial genome codes for 13 proteins, 2 rRNAs, and 22 tRNA genes • The 5 Kbp Common Deletion is believed to arise as the result of oxidative stress and is associated with age, diabetes and sun exposure • It accumulates in metabolically active slow turnover tissues such as muscle and nerve

3. Human Mitochondrial Genome Deletion occur Continue…

4. PCR conditions to select for deleted mitochondria: Polymerase doesn’t have time to replicate the normal 5kB piece Normal mitochondria MT1A 5kB MT2 MT3 __________________________________________ _____--------------------5 min ---------------------____ Deleted mitochondria MT1A 353 bp MT3 -------0.3 min-------- | \|/ MT1A 303 bp MT2 ------0.3 min-----

5. Our Goal!!! After the cells being exposed to the UV light, we wanted to find out the part of the DNA has been deleted in the Mitochondria DNA.

6. PCR machine Electrophoresis Centrifuge Materials

7. SV40 immortalized human keratinocyte line 22 at about 35% confluence were irradiated from the bottom with an FS20 lamp at a distance of 14 inches for 4 minutes (5.7 mJ/cm2/min). • 24 hours later, total DNA was prepared from the cells and used as a PCR template for deletion analysis. Methods Irradiation of Cultured Epithelial Cells

8. Continue… Method for Isolating DNA • Mix the NHEK cells with 200 µL of Pbs, to wash the cells • Add 20 µL of protein enzyme, to break up proteins • Add 4 µL of RNase, to break up RNA • Add 200 µL of buffer AL, to break down other substances • Incubate at 70°C for 10 min • Add 200 µL ethanol, to decrease the solubility of DNA • Then pipette the solution into the spin column & spin it for 1 min in a centrifuge • Add buffer AL1, to wash away the protein & salt • Centrifuge for 1 min, discard the column and add another column • add buffer AL2, to wash another time, then centrifuge for 3 min • Transfer in to a 1.5 ml tube • Add 50 µL of AE solution, which elute the DNA • Add 25 µL of AE solution to help more DNA to pass through

9. Continue… Electrophoresis • Took out four µL tubes & one 1.5 mL tubes • We took out 5 µL of cells from the original tubes and pipette them into the µL tubes • We label the 1.5 mL tubes as mixture which contains: - 8.8 µL of DNTP, - 22 µL of PCR buffer with MgCl2 - 4.4 µL of the primers that we need - 156.2 µL of sterile water • Put 2.2 µL of polymerase which has to be pipette at last because it reacts fast • Vortex the mixture • Add 45 µL of solution from the mixture tube into the µL tubes that contain cells • At last, put them into the PCR machine and we choose the program MT deletion After 1 and a half hour…. • Mix DNA with the dye • Pipette them into the gel • pipette the PCR marker at last • Then run the gel.

10. Uv &As Uv &As PCR Marker PCR Marker Con Uv As Con Uv As 1000 750 500 300 150 50 G2 – primer ML1 & MR4 G1- primer ML3 &MR5 Result

11. Further Research! • Clone the DNA • Sequence the DNA • Find out the sequence of the DNA that is being deleted

12. References • http://www.wou.edu/las/natsci_math/biology/boomer/waksman/WAKWORKS/workshop2/studentpjs/drummharvey/electrophresis.jpg • http://forestry.msu.edu/hardwood/Equipment%20Details/Flexigene%20PCR%20Machine.jpg • http://www.espchemicals.com/Images/group/1033.jpg

13. Acknowledgement • Dr. Mark Steinberg • Julia Wu • Bor Jang Hwang • Dr. Sat • Harlem Children Society • MSKCC • And Thank You!!!