DNA detection means determining if a specific DNA sequence is present in a sample.

This can be used to detect if a given species of microorganism is present in a sample, and is therefore a widely used diagnostics technique to see if someone is infected with a virus.

You could of course do full DNA Sequencing to see everything that is there, but since it is as a more generic procedure, sequencing is more expensive and slow.

The alternative is to use a DNA amplification technique.

Also known as: Quantitative PCR (qPCR).

Like PCR, but the amplification machine measures the concentration of DNA at each step.

This describes one possible concentration detection method with fluorescent molecules that only become fluorescent when the DNA is double stranded (SYBR Green)

This allows you to predict the exact initial concentration by extrapolating the exponential curve backwards.

TODO: vs non-real-time PCR. Why can't you just divide by 2 for every heating step to reach back the original concentration? Likely the reaction reach saturation at an unknown step.

TODO: vs non-real-time PCR in medical diagnostics: do you really need to know concentration for diagnostics? Isn't it enough to know if the virus is present or not?

At the time of the experiment, Illumina equipment was cheaper per base pair and dominates the human genome sequencing market, but it required a much higher initial investment for the equipment (TODO how much).

The reusable Nanopore device costs just about 500 dollars, and about 500 dollars (50 unit volume) for the single usage flow cell which can decode up to 30 billion base pairs, which is about 10 human genomes 1x! Note that 1x is basically useless for one of the most important of all applications of sequencing: detection of single-nucleotide polymorphisms, since the error rate would be too high to base clinical decisions on.

Compare that to Illumina which is currently doing about an 1000 dollar human genome at 30x, and a bit less errors per base pair (TODO how much).

Other advantages of the MinION over Illumina which didn't really matter to this particular experiment are:

After filtration, all DNA should present in the filter, so we cut the paper up with scissors and put the pieces into an Eppendorf: Video 1. "Cutting vacuum pump filter and placing it in Eppendorf".

Now that we had the DNA in Eppendorfs, we were ready to continue the purification in a simpler and more standardized lab pipeline fashion.

First we added some small specialized beads and chemicals to the water and shook them Eppendorfs hard in a Scientific Industries Inc. Vortex-Genie 2 machine to break the cell and free the DNA.

Once that was done, we added several reagents which split the solution into two phases: one containing the DNA and the other not. We would then pipette the phase with the DNA out to the next Eppendorf, and continue the process.

In one step for example, the DNA was present as a white precipitate at the bottom of the tube, and we threw away the supernatant liquid: Figure 1. "White precipitate formed with Qiagen DNeasy PowerWater Kit".

At various stages, centrifuging was also necessary. Much like the previous vacuum pump step, this adds extra gravity to speed up the separation of phases with different molecular masses.

In our case, we used a VWR Micro Star 17 microcentrifuge for those steps:

Then, when we had finally finished all the purification steps, we measured the quantity of DNA with a Biochrom SimpliNano spectrophotometer to check that the purification went well:

And because the readings were good, we put it in our -20 C fridge to preserve it until the second day of the workshop and called it a day:

One cool thing we did in this procedure was to use magnetic separation with magnetic beads to further concentrate the DNA: Figure 1. "GE MagRack 6 pipetting.".

The beads are coated to stick to the DNA, which allows us to easily extract the DNA from the rest of the solution. This is cool, but bio people are borderline obsessed by those beads! Go figure!

Then we prepared the DNA for sequencing with the Oxford Nanopore specific part: Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit.

Here some of the steps required a bit more of vortexing for mixing the reagents, and for this we used the VELP Scientifica WIZARD IR Infrared Vortex Mixer which appears to be quicker to use and not as strong as the Vortex Genie 2 previously used to break up the cells:

After all that was done, the DNA of the several 400 ml water bottles and hours of hard purification labour were contained in one single Eppendorf!

www.qiagen.com/gb/products/discovery-and-translational-research/dna-rna-purification/dna-purification/microbial-dna/dneasy-powerwater-kit (archive) Here is its documentation: www.qiagen.com/gb/resources/download.aspx?id=bb731482-874b-4241-8cf4-c15054e3a4bf&lang=en (archive).

Manual archive: web.archive.org/web/20190911111136/https://www.qiagen.com/gb/resources/download.aspx?id=bb731482-874b-4241-8cf4-c15054e3a4bf&lang=en

Kit to extract clean DNA from water.

www.velp.com/en/products/lines/3/family/44/vortex_mixers/65/wizard_ir_infrared_vortex_mixer (archive).

www.gelifesciences.com/en/us/shop/protein-analysis/protein-sample-preparation/protein-enrichment/magrack-6-p-05761 (archive).

Incredible that there hasn't been a Nobel Prize for it as of 2022, e.g. as mentioned at: theconversation.com/no-nobel-but-epigenetics-finally-gets-the-recognition-it-deserves-18970

Some old dudes getting another prize in 2016: www.cuimc.columbia.edu/news/pioneers-epigenetics-awarded-horwitz-prize

They are actually inheritable! But alleles are rare: www.ncbi.nlm.nih.gov/pmc/articles/PMC5559844

Sequence of genes under a single promoter. For an example, see E. Coli K-12 MG1655 operon thrLABC.

A single operon may produce multiple different transcription units depending on certain conditions, see: operon vs transcription unit.

Current Wikipedia seems to say that this refers specifically to cells taking up DNA from other dead cells as in the Avery-MacLeod-McCarty experiment, excluding other types of horizontal gene transfer like bacterial conjugation

The term is sometimes just used a synonym for horizontal gene transfer in general it seems however.