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

By: Jing Jing HeMentor: 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_______________________________________________--------------------5 min ---------------------____

Deleted mitochondriaMT1A 353 bp MT3

-------0.3 min-------- |

\|/MT1A 303 bp MT2------0.3 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!!!

MaterialsElectrophoresis 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/cm2/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 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

Continue…

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.

Result

1000

750

500

300

150

50

G1- primer ML3 &MR5

G2 – primer ML1 & MR4

Con Uv As

Uv

&AsPCR Marker Con Uv As

Uv

&AsPCR Marker

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

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

Acknowledgement

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