Open Science Hardware to Open Source Classic Methods of DNA Damage Detection: the Cheek Cell Chip

Rachel Aronoff: AGiR! CSO & Hackuarium President

Open Science Hardware

Action for Genomic integrity through Research!

Once we learned that the BRCA genes are really a ‘loss of function’ for correct homologous recombination to repair DNA damage, and that these being mutated increases risk for not only breast cancer in women, but prostate cancer in men (and many other cancers!), the idea that avoiding DNA damage could be a very wise seemed worth spreading.

Prevention is better than a cure!

AGiR! was founded in 2013, aiming to provide information and promote research!

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We have at least 100 genes to repair DNA damage, but some repair pathways can make things worse!

‘Open Sourcing’ classic methods of DNA damage detection

Micronucleus & Comet Assays

both methods used for

risk analyses since

around 1970…

Shows

big pieces of

broken off

chromosomes

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Shows

overall levels

of double stranded breaks

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https://www.sigmaaldrich.com/CH/de/technical-documents/technical-article/research-and-disease-areas/cancer-research/comet-assay

The comet assay is frequently done on blood, but inner

cheek cells

are also good!

DNA has a negative charge, so

goes toward the positive

pole in an electric field.

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This makes the comet tail!, which is visible through use of various fluorescence stains.

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(SYBR Safe, is the one we generally use, although it is expensive and not really safe…

(but better than alternatives)

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nov2019

cheek cells in agarose ‘crềpe’

Use of Open Science Hardware

During GOSH2018, the Gathering for Open Science Hardware in 2018 in Shenzen, learned about the:

• OpenFlexure Microscope
• developed in the UK (Bowman lab)
• 3D printed
• high-resolution version for epifluorescence (needed for comets)
• can image whole sample as a panorama

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First build

with motors & motor board

Towards the Cheek Cell Chip

First build with ordinary 10x objective (plan)

and Raspberry Pi camera

• Could just see nuclei, but not tails of comets
• Fine for micronuclei, which can even be scored in physiological conditions, but more work was needed…
• THEN came the PANDEMIC!

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Now, GOSH is helping fund further advances.

YAY!

FLUOTAR objectives

&

Higher Res cameras

Will compare the Delta format and the latest version of the OpenFlexure, with these better elements than in our first build, to see if comet tails will also be visible.

Then, for the best system in this context, can make a third build with a modified stage holder for millifluidics to do: first, imaging of cells for micronuclei, and then, treatments and e- for comets, and then image it all again.

That will provide good measurements of the level of DNA damage in each cell of the sample.

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expensive optics for 3D printed equipment, to me…

but, going for it!

To obtain:

• frequency of micronuclei/1000 cells
• ratio of comet tail fluorescence to that found in the remaining nucleoid

Lorem ipsum dolor sit amet.

30june22

Delta stage

OpenFlexure

v7 alpha2

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RaspberryPI

12.3Mpixel

x2

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Sourcing the FLUOTAR objectives is challenging.

Getting all the bits and pieces (big O- rings, fluor filters, etc.)

Workshops - planning for July and September.

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Thanks to GOSH, also for helping us host Mitch Altman

rachel@hackuarium.ch

@AGIRgenomes

@genomicintegrity.org

@hackuarium