CometAssayCometAssay™™

Society of Toxicology Exhibitor Hosted Session Trevigen®

Sandra Woodgate, PhD Thomas Uveges, PhD

BackgroundBackground

• The Comet Assay is a single cell gel electrophoresis assay for evaluating DNA damage in cells•• Based on denatured, cleaved DNA fragments migrating out Based on denatured, cleaved DNA fragments migrating out

of the cell when a current is applied whereas undamaged of the cell when a current is applied whereas undamaged DNA migrates slower DNA migrates slower

• Applications •• Study cellular response to DNA damageStudy cellular response to DNA damage•• Screening assay for inhibitors to DNA repair (cancer Screening assay for inhibitors to DNA repair (cancer

therapy)therapy)•• Testing chemicals for toxicityTesting chemicals for toxicity

Assay OverviewAssay Overview

1. Cells mixed with low melting agarose at 37°C (LM Agarose)

2. Immobilize cells on CometSlide™

3. Treat cells with Lysis Solution (removes membranes and histonesfrom the DNA)

4. Samples treated with alkali (unwinds and denatures DNA)

5. Alkaline electrophoresis performed (reveals DNA breaks)

6. Samples stained with intercalating dye and visualized by epifluorescence microscopy.

1.

2.

3. & 4.

5.

6.

Sybr® Green I

DNA migration out of nucleoid

Comet DefinitionsComet Definitions

• Percent DNA in the Tail•• The integrated tail intensity x 100 divided by the total The integrated tail intensity x 100 divided by the total

integrated cell intensity for a normalized measure of the integrated cell intensity for a normalized measure of the percent of total cell DNA found in the tailpercent of total cell DNA found in the tail

• Tail Moment•• The product of distance and normalized intensity integrated The product of distance and normalized intensity integrated

over the tail length, over the tail length, ΣΣ (L(Lxx •• % % DNADNAxx))•• a damage measure combining the amount of DNA in the tail a damage measure combining the amount of DNA in the tail

with the distance of migration (severity of damage)with the distance of migration (severity of damage)

Alkaline Electrophoresis ConditionsAlkaline Electrophoresis Conditions

• Standard Procedures•• Singh et al. 1988Singh et al. 1988•• Olive and Olive and BanathBanath, 2006, 2006• http://cometassay.com

• Fresh Alkaline Buffer and large horizontal tank•• Cold 1 Cold 1 mMmM EDTA and 300 EDTA and 300 mMmM NaOHNaOH, pH>13, pH>13•• Add buffer so barely covering slidesAdd buffer so barely covering slides•• Electrophoresis for 20Electrophoresis for 20--40 min40 min

–– 0.7 V/cm or 1 V/cm at 300 0.7 V/cm or 1 V/cm at 300 mAmA

ElectrophoresisElectrophoresis

Variables: Alkaline ElectrophoresisVariables: Alkaline Electrophoresis

• Electrode Distance and Voltage• 1 volt/cm (300 mA) is recommended. Amperage problem with

some tanks.

• Buffer Height• More results in less electrophoresis.

• Buffer Temperature• Alkali solutions are not buffered and great variations occur,

recirculation and/or cold electrophoresis works best.

• Electrophoresis Time•• Longer tails with longer electrophoresis time.Longer tails with longer electrophoresis time.

• Buffer Effects on Amperage• Different comet shapes and tail lengths by changing

pH of electrophoresis solutions• High buffer concentrations affect amperage

• Position equal distance between electrodes• To minimize well to well variation

• Slide Orientation•• Movement during electrophoresis affects Movement during electrophoresis affects

measurementsmeasurements

Variables: Alkaline ElectrophoresisVariables: Alkaline Electrophoresis

Control CellsControl Cells

•• Control CellsControl Cells to standardize and compare to standardize and compare electrophoresis methods between users and electrophoresis methods between users and laboratories.laboratories.•• Four unsynchronized suspension cell Four unsynchronized suspension cell

preparations with incremental increases of preparations with incremental increases of DNA damageDNA damage

•• Designed to be used when performing alkaline Designed to be used when performing alkaline electrophoresiselectrophoresis

•• Designed for long term storageDesigned for long term storage

Control Cells: % DNA in TailControl Cells: % DNA in Tail

•• Four statistically distinct populationsFour statistically distinct populations

-10

10

30

50

70

90

CCO CC1 CC2 CC3

Control Cells

% D

NA

in T

ail

% DNA by Etoposide n Mean SD SE 75% CI of Mean Median IQR 75% CI of MedianCCO 50 5.757 7.7270 1.0928 4.485 to 7.029 1.640 8.925 1.290 to 2.230CC1 50 28.374 14.0080 1.9810 26.068 to 30.680 28.990 20.313 25.180 to 31.840CC2 50 39.736 21.8164 3.0853 36.144 to 43.328 37.050 32.183 27.790 to 44.630CC3 50 56.800 23.5893 3.3360 52.916 to 60.683 51.905 40.240 45.460 to 64.390

Electrophoresis Tanks Electrophoresis Tanks

• In-house•• BioBio--Rad DNA SubRad DNA Sub--CellCell

–– older modelolder model

•• 16 cm x 30 cm (L)16 cm x 30 cm (L)

• Developed for Comet•• Cleaver ScientificCleaver Scientific

•• 28 cm x 25 cm (L)28 cm x 25 cm (L)

Optimize Running ConditionsOptimize Running Conditions

* Not possible to always adjust to 300 mA by buffer height• 0.4 cm with 300 mM NaOH is > 500 mA

– 40′ at 0.7 V/cm (300 mA) using 300 mM NaOH– 20′ at 1 V/cm (300 mA) using 150 mM NaOH

Bio-Rad CleaverRunning Conditions~1L Alk Buffer pH>13 20′ at 1V/cm (300 mA)

300 mM 150 mM*

Control Cells (5 replicates)COC1C2C3

% DNA (S.D.)6.7 (0.86)

32.4 (2.03)44.08 (1.63)60.6 (3.62)

% DNA (S.D.)9.46 (1.47)31.2 (2.45)43.4 (2.41)66.2 (0.9)

Alkaline Buffer ConcentrationAlkaline Buffer ConcentrationpH [OH-] (M)10 10-4

11 10-3

12 10 mM13 100 mM14 1M

• 300 mM NaOH, pH >13•• typically recommended to maintain strand typically recommended to maintain strand denaturationdenaturation•• Some users unable to run at 1V/cm due to high amperageSome users unable to run at 1V/cm due to high amperage

• pH 13 is achieved at concentrations > 100 mM NaOH

Vary Slide Position on PlatformVary Slide Position on Platform

• Differences in Comet shape• Position dependent• Faster migration near cathode (length)

Anode

# Counted TM % DNA Length

C2 - A C

111111

9.0414.8

43.3651.27

38.454.2

C3 - A C

8494

15.4923.03

59.3867.69

46.7561.60

30 cm electrode distance/300 mM NaOH

CathodeC3 C3

BioRad Unit

- +

Designed Prototype TankDesigned Prototype Tank• Amperage and Temperature• [NaOH] pH>13• Electrode distance and Buffer height • Slide Position

1 2 3 4 5 6 7 8 9 10 11 12Electrode positions Electrode positions

Platform

Amperage Amperage

• At constant volts, Amperage increases with•• Electrode DistanceElectrode Distance [[NaOHNaOH]]•• TemperatureTemperature Buffer HeightBuffer Height

• 300 mM NaOH problematic for 400 mA powerpacks•• Electrophoresis at 4Electrophoresis at 4°°C is needed keep amperage <400 C is needed keep amperage <400 mAmA))

150 mM NaOH - 4°C150 mM NaOH - 24°C300 mM NaOH - 4°C300 mM NaOH - 24°C

Electrode Distance (0.4 cm buffer)

100

200

300

400

500

11 13 15 17 19 21

Electrode Distance (cm)

mA

mps

(c

onst

ant 1

V/cm

)

Buffer Height (16 cm distance)

100

200

300

400

500

0.4 0.6 0.8 1Buffer Height (cm)

mA

mps

(c

onst

ant 1

V/cm

)

V=IR

[[NaOHNaOH] pH >13] pH >13

30 min/21 cm30 min/21 cm1V/cm, constant buffer height1V/cm, constant buffer height

C1 C3 C1 C3 C1 C3 150 mM 200 mM 300 mM

• Length increases with lower [NaOH]

• Better sensitivity w/ 150-200 mM NaOH

Comet Length vs NaOH

0

20

40

60

80

150 200 250 300

mM NaOH

Leng

th C1C3

Electrode Distance Electrode Distance –– Buffer HeightBuffer Height

• Adjusting to 300 mA using buffer height reduces Comet length as electrode distances are decreased•• Buffer height >0.5 cm reduces lengthBuffer height >0.5 cm reduces length•• Electrode distance < 18 cm reduces Electrode distance < 18 cm reduces

lengthlengthElectrophoresis - 1V/cm (300 mA), 150 mM NaOH

Elect Dist: 13 cm 16 cm 18 cm 21 cm

CO C1 C2 C3 CO C1 C2 C3CO C1 C2 C3 CO C1 C2 C3 CO C1 C2 C3

Length/Buffer Height vs Electrode Distance

0

20

40

60

80

100

120

13 15 17 19 21

Electrode Distance (cm)

C2

Leng

th

Buf

fer H

eigh

t (m

icro

n)

Buffer Height

C2 Length

Control Buffer Height Control Buffer Height -- OverlayOverlay

Low Buffer

010203040506070

1 2 3

Well Position

% D

NA

COC1C2C3

High Buffer

010203040506070

1 2 3

Well Position

% D

NA

COC1C2C3

High buffer- overlay

010203040506070

1 2 3

Well Position

% D

NA

C0C1C2C2

• Piece of plastic placed over slides• Reduces variation due to buffer height

21 cm electrode distance/300 mM NaOH

0.4 cm buffer height 1 cm buffer height Overlay

Vary Slide Position on PlatformVary Slide Position on Platform

• Stacking Slides increases Variation •• ~50% Voltage Drop across slide platform~50% Voltage Drop across slide platformTop

Bottom

CO C1 C2 C3

CO C1 C2 C3

# Counted TM % DNA Length

CO TB

208321

0.540.02

3.920.96

3.71.2

C1 TB

259293

3.070.46

21.485.12

18.84.8

C2 TB

135139

4.840.68

29.235.96

235.4

C3 TB

139111

7.480.39

40.354.73

29.24

29 cm electrode distance/300 mM NaOH

Model TankModel Tank• 21 cm in length• Run at 1V/cm, ~300 mA for 30-40 minutes

• Use with most laboratory power supplies• No well to well variation

• Overlay to maintain 0.4-0.5 cm buffer height• 1L of buffer, 150-300 mM NaOH pH >13

• Platform accommodates 1 row of slides• Maintain slide position• Use 2, 3, 20 and 96 well slide formats• Easy removal and placement of slides

• Maintain buffer temperature ~4°C• Ceramic plate and cooling chamber Coming Soon!

Exploded DiagramExploded Diagram

Overlay

Interlocking Safety Lid

Ceramic

2/20 Well Tray

Water Chamber Ports Coming Soon!

SamplesSamples

• 2/20 well Tray• Ten 2-well slides• Five 20-well slides

• 96 well Tray• 3 slides - 288 samples

96 Well Tray

Coming Soon!

Alkaline pH >13Alkaline pH >13

• Good sensitivity in 30′ w/ 200 mM NaOH (< 300 mA)• Or 40′ w/ 300 mM NaOH (>300 mA)• Achieved Specifications for Control Cells

% DNA in Tail200 mM NaOH

220 mA for 30′

% DNA in Tail300 mM NaOH

380 mA for 40′

CO 7.21 7.29

C1 24.57 27.64

C2 40.39 40.71

C3 56.63 55.96

1 V/cm using 2 well slides

96 well slide96 well slideMinimal well to well variation with Slide Overlay

010203040506070

1 2 3 4 5 6 7 8 9 10 11 12

Well Position (cathode -> anode)

% D

NA

in T

ail

COC1C2C3

0102030405060

1 2 3 4 5 6 7 8 9 10 11 12

Well Position

Leng

th

COC1C2C3

Buffer Height Maintained with Slide OverlayBuffer Height Maintained with Slide Overlay

• Buffer height increases without slide overlay• % DNA in Tail Decreases• Length Decreases

% DNA Length+ Overlay - Overlay + Overlay - Overlay

CO 11.76 6.2 12.38 4.67C1 29.76 13.93 31 10.5C2 43.47 26.09 39.08 19.08C3 59.94 35.25 48.17 24.7

Comet Parameters Comet Parameters –– Slide FormatSlide Format

• 30 'electrophoresis for 2/20 well slides

• 40' electrophoresis for 96 well slides

TM % DNA LengthCO 2 well

20 well96 well

1.421.641.62

7.217.1011.76

151712

C1 2 well20 well96 well

4.626.275.11

24.5728.6929.76

363931

C2 2 well20 well96 well

10.4510.969.07

40.3942.9943.47

504739

C3 2 well20 well96 well

16.3915.9816.02

56.6355.7159.94

555448 1V/cm, 200 mM > pH 13

Modified Comet Assay Modified Comet Assay –– DNA DamageDNA Damage

1. Cells Mixed with LM Agarose• Spread 40 µl

2. Treat cells with Lysis Solution• 1 hr at 4°C

3. Equilibrate with Enzyme Buffer• 3x 10′ at RT

4. Treat with Enzyme• 45′ at 37°• Hydrophobic coverslip

5. Treat with Alkali unwind6. Alkaline electrophoresis7. Samples stained and visualized

• Type of Damage• UV Damage• Oxidative Damage• AP sites

• DNA Repair Enzymes• Lesion Specific• Bacterial enzymes

Repair EnzymesRepair Enzymes

• FPG • glycosylase for oxidized purines with associated AP lyase• 8-oxoGua, FaPyGua, FaPyAde

– other ring-opened purines

• ENDO III• glycosylase for oxidized pyrimidines with associated AP lyase

– including thymine glycol and uracil glycol

• hOGG1• glycosylase for oxidized purines with cleavage of phophodiester

bond through Schiff base chemistry• 8-oxoGua, FaPyGua

Oxidative Damage with KBrOOxidative Damage with KBrO33

• FPG showed a dramatic increase in breaks for Healthy cells compared to hOGG1 and ENDOIII

• FPG and hOGG1 showed a similar increase in breaks with Treated Cells compared to Healthy cells but not for ENDOIII.

• hOGG1 more specific for oxidative damage with KBrO3• Mutagenesis 2006

% DNA in TailNo Enzyme FPG hOGG1 ENDOIII

Healthy Cells 4.3 21.3 * 7.7 8.2 Treated Cells 5.0 33.4 * 13.0* 6.0

* significant

Monitor DNA RecoveryMonitor DNA Recovery

• H2O2 treatment - high level of damage

• 2 hr recovery - reduction in all comet parameters

TM % DNA LengthUntreated 1.0 3.4 22

H202 Treated 38.6 87.3 71

1 hr Recovery 7.4 25.5 55.3

2 hr Recovery 2.7 9.4 28

U T 1hr 2 hr

Alkaline Comet

Electrophoresis BuffersElectrophoresis Buffers

• Alkaline Buffer• Droplet comet shape

•• Greater sensitivityGreater sensitivity–– ssss-- and and dsds--breaks breaks –– alkalialkali--labile sites (AP sites)labile sites (AP sites)

• Neutral Buffer (TBE)• Elongated comet shape

• Reduced sensitivity – due to DNA renaturation– Detection of ds-breaks

Treat with PI3Kinase inhibitor (Wortmannin)

DNA DAMAGE (Etoposide)

Activation of DNA-PK pathway for NHEJ repair

Inhibit DNA-PK pathwayRecovery from DNA damage

• Unable to repair DS breaks by DNA PK mediated NHEJ• Cell unable to pass cell cycle checkpoints• If level of damage high enough and/or unable to repair damage

Cell death occurs

DNA Repair of Double Strand BreaksDNA Repair of Double Strand Breaks

Neutral CometNeutral Comet

• Double-strand break repair in 1 hr• PI3 kinase inhibitor Wortmannin inhibits ds repair• Damage levels remain same in presence of inhibitor

# TM %DNA Length95 0.98 7.66 8

138 0.95 8.34 1074 2.23 14.15 19

114 2.19 15.57 1988 0.63 6.94 1078 0.34 4.63 695 1.94 14.09 1964 1.93 11.03 16

Untreated

Damaged

Recovery

Inhibitor

SummarySummary• Control Alkaline Electrophoresis

• Optimized Electrophoresis System• CometAssay™ Control Cells• To monitor DNA Damage and Repair

Patent Pending

Trevigen, Inc.1.800.873.8443info@trevigen.comwww.trevigen.com

© 2008 Trevigen, Inc. Trevigen is a registered trademark and CometAssay and CometSlide are trademarks of Trevigen, Inc.