- (gentle piano music)
- Jacques, thank you so much for inviting me to your home.
- Thanks so much for coming.
We got so much information during our trip,
I thought it would be nice to go over
some of the interesting pieces we had to cut out.
- Absolutely, a debrief would be great.
There was just so much good stuff,
if we had to include everything,
the episode would be an hour long.
- This is meant to be a supplement to the main episode
in Adventures in Genomics.
- So if you haven't checked out our main episode,
please be sure to check that out first.
The link will be in the description below.
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- Now that we're back from Christina Warriner's lab
at the University of Oklahoma,
what was the highlight for you?
- You know, I loved everything about it,
but what really stuck out in my mind
was the picture of the galaxy that she showed.
- [Jacques] It is amazing when you see those microbes
it really does look like a galaxy.
Almost feel I could fly through it.
- And to think that the number of microbes
in and on our body exceeds the number of stars
in over 300 galaxies.
- [Jacques] On those pictures, every point of light
is a microbe and you feel like you can reach out
and touch them and then you realize,
these things are thousands of years old.
- It's almost like time traveling.
- It really is, it really is.
And who knows where this journey is going to lead.
But one thing you can be sure of,
is it's going to be a fascinating journey.
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- [Jacques] I love this electron microscope image
of the calculus.
It shows a huge amount of information.
- [Swati] Absolutely, you can really see how the calculus
was formed in layers over the life of the individual.
- You know the exact mechanism of calculus deposition
is still unknown, but we do know that the process
is appositional.
That means that there are alternating bands
of gram-positive and gram-negative bacteria.
- And you can see the layers in this image.
If you look closely, you can see individual
filamentous bacteria oriented perpendicular
to the tooth surface.
So why doesn't the DNA deteriorate when the bacteria die?
- That's a really good question.
As the calcium phosphate in the saliva
starts to precipitate, it builds up crystals
that prohibit nutrients from going from the outside
of the bio-form to the inside.
And then, the bacteria in the inside,
they start starving and they start to die,
but their iron pumps stop working and they start
to calcify then from the inside out.
- So it's a two-step process.
I mean, the bacteria calcify from the inside out
and then from the outside in.
- That's exactly right.
This dual process is very important for DNA preservation
because the enzymes don't have time to break down the DNA
and also the mineral that actually forms
is hydroxyapatite, which is excellent for binding the DNA
and for protecting it.
- Absolutely, and you know, we were focusing
on the microbes, but the calculus also captures
microfossils like bits of food.
- You know, they are fascinating
because they actually tell you how the person lived.
- [Swati] Here we have microcharcoal from smoke
or burnt material and a burnt pollen grain.
When you see it, you can imagine and almost smell
the fire and the birch forest.
- It is very evocative.
Plant and animal materials are very hard
to classify visually.
And here again, they're starting to use sequencing
to identify genetic sequences consistent
with specific foods like pork, wheat, and sheep.
- You know, just imagine getting all this information
from a tiny little piece of calculus.
It's truly adding a whole new dimension
to this ancient field of paleontology.
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- Jacques, I was amazed at how calculus
can act as a capsule to preserve the DNA.
- You know, Christina mentioned
that both bones and teeth are porous
and that the microbes can use those channels
to infiltrate and decompose the bone after death.
- You know, those channels are needed
to heal and grow bones, and bone is constantly
being remodeled, so even dense bones need a means
of profusion.
- Well, you know, calculus is different.
Even though it's the same material as bones and teeth,
it's formed from calcium phosphate, it's a precipitate
and it actually encapsulates whatever is
in the dental plaque at that time.
- So there's no channels and it's not porous.
- That's absolutely right, and another important point,
calcium phosphate is in the form of hydroxyapatite,
and that actually binds the DNA which may actually help
to preserve it.
- So if the DNA is bound to a substrate,
it will be protected from degradation by nucleases?
- You know, that's right, and interestingly enough
when, in the old time when I was still working in the lab,
hydroxyapatite was pretty commonly used
to, and columns, to purify the DNA.
You know, calculus also forms during your lifetime,
so the microbes are encapsulated
while they're still alive and fresh.
- Yeah, it skips the whole pesky death and decay process.
But calcification actually is a really complex process
and it's part of the reason why the material
is so well preserved.
And we'll cover that much more in another section.
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- [Swati] Jacques, this is by far one
of my most favorite images.
You can just see all the microbes in the calculus.
- [Jacques] They used Hoechst stain to show the abundant
in situ double stranded DNA in decalcified calculus.
- [Swati] Now, when you compare the modern
and thousand-year-old ancient DNA slides,
you can just see how remarkable the preservation is.
- [Jacques] Every pinpoint is an individual microbe,
but if you look really closely, and it is most apparent
on the modern DNA, you see some DNA outside the cells.
- [Swati] So, is that extra-cellular DNA?
- That's exactly right.
That is DNA from the microbes.
It is part of the three-dimensional scaffolds
the cell need to locate and then to orientate themselves.
- So the matrix is also thought to act
as a nucleation point to start the calcium phosphate
crystal formation.
- Yes, and of course the calcium phosphate crystals
are the hydroxyapatite that binds the DNA.
The microbes need the matrix and that is one of the reasons
so few microbes can usually be cultured in the lab
and now next generation sequencing
is making a really big difference in the studying
of dental microbes.
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- You know, Jacques, the clean rooms were very impressive
and it is one of the most advanced laboratories
in the country.
- There's a lot more to it than you see in the video.
When the people are not working in the laboratories,
strong UV lamps kill the microbes
and they destroy the DNA.
- You know, I remember, we actually had to arrange
our schedule around the lab protocols.
- That's true.
The movement between the labs is tightly controlled.
- [Swati] Yes, and you know there are several layers,
each getting progressively cleaner.
- [Jacques] You know, to go into the cleanest labs
would have taken a lot more precautions
than the ones we took.
- [Swati] You do not wanna contaminate the ancient samples
with our modern DNA.
- It's not just our DNA.
Microbial contamination is a huge problem.
The polymerases that are used for the amplification process
are derived from microbes and they would preferentially
amplify microbial DNA to the exclusion of human sequences.
- We know how important it is to contain
sample contamination, but we will cover that
in another section.
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- The DNA fragment size was an interesting story to me.
The short fragment size used to be a huge problem
with PCR and Sanger sequencing.
Remember PCR will preferentially amplify shorter pieces,
so that actually makes the problem worse.
- You know, for next-gen sequencing,
it actually makes it easier because they do not have to do
the fragmentation step when they're doing library prep.
- You know, it's one of those applicattions
that was just not possible before next-gen sequencing.
- [Swati] You know, and Christina mentioned
that they typically see fragments of 50 to 90 bases long
depending on how well-preserved a sample is.
- [Jacques] You know, with very old samples,
say in the order of hundreds of thousands of years,
the size can drop down to 25 bases.
- And that's still long enough to get, you know,
useful information.
- Yeah, and apparently the oldest authentic ancient
DNA that has ever been studied
is about 700,000 years old and it was recovered
from a horse in the permafrost.
- You know, I really wonder how long that record will stand.
- Probably not very long, the way things are going.
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- So the samples are heavily contaminated
when they enter the lab.
- Freshly dug up.
Well, the good news is that calculus
does not have natural channels and pores
so external bacteria cannot penetrate it,
especially once it's calcified in your mouth.
- The thought still just creeps me out.
But it does mean that they only need to clean the surface
to remove environmental contaminants.
- Exactly.
They just soak it in EDTA to decalcify the outer layer,
and then they agitate it vigorously to shake off dirt
and environmental contaminants.
- So what they do is they save the wash solution
as a control so they can compare the sample
with the contamination.
- Yes.
Some microbes can be used as controls
because, for example, acetobacteria,
it's very common in the soil, but it's very rare
in the calculus.
So if you see acetobacteria,
you know that you've got a contamination problem.
- Absolutely, and as the technology's getting
increasingly sensitive, it is crucial to have
good experimental designs and controls.
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- The simplicity of the sample prep really surprised me.
I expected they would be grinding the samples
under liquid nitrogen or something equally heroic.
- I agree, I was actually expecting the same.
But you know, Christina did mention
that they tried many many methods
until they found one that actually worked.
- They list their results from the various methods
in their nitrogenetics paper from 2014.
That paper has 100 pages of supplemental materials.
In the end, it turns out, the most successful
was also the simplest.
They simply tumble the calculus in EDTA
and then they add a little bit protein assay.
- [Swati] And then they use the silicon columns for clean-up
and from there, standard aluminum library prep
and sequencing.
- Their methods are very well described
in the paper I mentioned earlier.
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- Tannerella forsythia is a gram-negative member
of the bacteroidetes.
It's also associated with advanced periodontal disease
and has been reported in atherosclerotic lesions.
It is pretty amazing that they were actually able
to sequence this in 30-fold coverage.
- You know, just because the DNA was there,
it doesn't mean that the microbe was alive and active.
- That is a very good point.
In a fresh sample, you could simply sequence the RNA
and know if it's alive.
- And see what it was doing.
- Of course.
In this case they had to use mass spec
and they identified peptides belonging
to 10 forsythia proteins.
- That would confirm that it was alive
and making proteins at one stage.
Now, it is very powerful when you can combine sequencing
with such technologies such as mass spec.
- When you combine the two, you actually get a really great
insight into some of these ancient tissues.
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- Jacques, when you see the tooth,
it is just so strange to see that massive chunk of calculus.
- [Jacques] It's not intuitive at all,
and you really see it because it's usually cleaned off
by your dentist.
So if you see modern examples of heavy calculus
in a textbook, you can only be glad
that they actually do that.
- [Swati] You know, in the museum collections,
the calculus is actually scraped off
so anthropologists can see the characteristics
of the teeth.
- [Jacques] Now we know that what they throw away
is arguably the most interesting part of the sample.
- You know, and this sample was 4,000 years old,
and it makes you wonder what dental hygiene
was like back then.
- It depends a bit on the location.
You know, in some traditions they used sticks
or toothpicks and so on.
Whole books have been written on this subject.
And there's some evidence, actually,
that Neanderthals used toothpicks.
- You know, maybe this work will help
shed some light on that subject as well.
- We just never know what you'll discover next.
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