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

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 Introduction

Human Mitochondrial Genome Continue… Deletion occur

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

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. Our Goal!!!

Materials Electrophoresis PCR machine Centrifuge

Irradiation of Cultured Epithelial Cells 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/cm 2 /min). 24 hours later, total DNA was prepared from the cells and used as a PCR template for deletion analysis. Methods

Method for Isolating DNA  Mix the NHEK cells with 200 µ L of Pbs, to wash the cells  Add 20 µ L of protein enzyme, to break up proteins  Add 4 µ L of RNase, to break up RNA  Add 200 µ L of buffer AL, to break down other substances  Incubate at 70°C for 10 min  Add 200 µ L 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 µ L of AE solution, which elute the DNA  Add 25 µ L of AE solution to help more DNA to pass through Continue…

Electrophoresis  Took out four µ L tubes & one 1.5 mL tubes  We took out 5 µ L of cells from the original tubes and pipette them into the µ L tubes  We label the 1.5 mL tubes as mixture which contains: µ L of DNTP, - 22 µ L of PCR buffer with MgCl µ L of the primers that we need µ L of sterile water  Put 2.2 µ L of polymerase which has to be pipette at last because it reacts fast  Vortex the mixture  Add 45 µ L of solution from the mixture tube into the µ L 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.

Result G1- primer ML3 &MR5 G2 – primer ML1 & MR4 Con Uv As Uv &As PCR Marker Con Uv As Uv &As PCR Marker

Further Research!  Clone the DNA  Sequence the DNA  Find out the sequence of the DNA that is being deleted

References  KWORKS/workshop2/studentpjs/drummharvey/electrophresis.jpg  %20PCR%20Machine.jpg 

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