De extincting dinosaurs or at least extict birds

[Mega]

Just saw a film on TV that T-Rex bones were found which still contained few proteins like collagen. Then I read that this is very controversial, if that is not contamination.

Then I came across this...
http://canterburyheritage.blogspot.co.at/2009/01/extinct-moa-to-be-brought-back-to-life.html

The giant bird, Moa, is relatively new extict and there should be plenty of sources for DNA... Even eggs are said to be still there.


I assume there won't be any cells any more that are still alive...? Is it possible to purify the chromosomes, insert in a living cell? I assume that it would be near-to- impossible in a diy-setting, because the DNA will be damaged at least to some degree...


However, from the legal point of view, this should be perfectly legal (while inserting GFP into E.Coli is a capital crime)...



Any opinions on this?

[Alexey Zaytsev]

Certainly not today, maybe in 20-30-40 years, but I would not bet on this.
You can read the genome today, but how would you re-create it?
Re-assembling DNA extracted from bones/eggs is just out of question.
One way would be to find a close relative of the moa, and replace some
of its genes one by one. Does not sound too easy. Do we have a
reliable method of replacing large pieces of eukaryotic genome in vivo
already? How do you identify which parts are important and which are
not? The difference is almost certainly a lot more about regulation
than about changes in proteins. Even if we could do this, it would
still take ages to perform the modify-check cycles, and one could
always claim that the result is not really a moa, but a GM ostrich
made to look like one.
An other way - synthesize the whole genome. I'm pretty sure we can't
synthesize anything remotely as big as a chromosome, and even if we
could, there's a lot more than just DNA in a chromosome, and a lot
more then just the sequence that's carrying the information. If
developed, this technology would be really interesting, and also
really controversial (you could synthesize a human genome), but I
doubt that we will see it any time soon.

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[Andreas Sturm]

Stupid question,

On Antarctica there should have been dinosaurs (before the ice cap). May there still be frozen ones?

Maybe they have been exposed to warm temperatures for millennia, but not for eons, thus some DNA may still be there?

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[Andreas Sturm]

Found somtething (in German)
http://www.nationalgeographic.de/aktuelles/dinosaurier-in-der-antarktis

Antarctica was more Nord in these days, had a milder climate.

Another thing I read that near the coasts it was warmer, clearly.

Seems there might be a small chance that a dinosaur wandered from the coast into the cold inner land, died there, got frozen and is still there?

[A.J. DNA]

Heh... opinion.

So I've been extensively researching this topic for a little side project I am considering. It's worth noting that the group led by M Schweitzer and J Asara have written quite a few papers backing up their original findings. One important aspect is that proteins were not discovered... but peptides were. The crux of the matter lies with their discovery of peptides originating from histones in a hadrosaur fossil, which as you may recall bacteria do not possess. Of course, this does not rule out contamination, but it does eliminate the most likely culprit. Here's the DOI for that paper, where they do a fantastic job backing up their results with mass spec and immunohistochemical assays.:

DOI: 10.1016/j.bone.2012.10.010 (Let me know if you cant get it and I'll happily provide the pdf. Just shoot me an email.)

Another important consideration is that the peptides in question share significant homologies with avian peptides. This, I imagine, would be a tough feat for peptides from, say, a fungus, to pull off. Maybe an ancient moa-like bird used the fossil bed as a nesting site? So, you know, there is room for SOME doubt, if you wanted to be difficult. But these results would certainly hold up in court. Obviously, more work needs to be done, and more peptides discovered.

As you can probably tell by my post, I believe the results. The biggest question now is how common are these peptides? Does fossil preservation need to be exceptional, or can they be found in most fossils, provided you do control for contamination? This would be a big problem for any fossil that has already been dug up and is in a museum basement.

If the peptides are common enough, and whole protein sequences can be determined, it's simple enough to reverse-translate the genome. As has been said though, it's still a long way off.

~A.J.

[Ashley Heath]

Elsevier....! Unable to get the paper, would you contact me at ahe...@sial.com when you get a moment please?

[Andreas Sturm]

Is a peptide bond more stable than nucleotide bonds?

Put it the other way:

If the peptides can be conserved, can there be DNA remaining intact to some degree?

> Maybe an ancient moa-like bird used the fossil bed as a nesting site?

Therefore you would need to find another dinosaur bone, if it has the same peptide sequence, it would fit well.

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[Andreas Sturm]

Or it would show that Moa birds loved dinosaur bones as nesting sites... ^^

[A.J. DNA]

The reason that these results are so surprising is that people thought a snowball could survive in hell better than a peptide could survive the fossilization process. It has nothing to do with the stability of peptide or phosphodiester (nucleotide) bonds, which supposedly break down after a few thousand years. They hypothesize, as I understand it, that some special property of this particular fossilization of bone material was able to form a crystal matrix that protected some peptides from degrading. What's really cool is that not only are there peptides, there are fossilzed osteocytes (bone cells) in there as well.

So basically, if they can find peptides, there is no reason they cannot find nucleic acid buried in the crystal matrix. The problem is that both are likely to be very short sequences, maybe 10 or 20 units long. This isn't a problem for peptides, because with 10-20 amino acids it's much easier to identify what protein it belonged to using a mass spec. With a DNA molecule, 10-20 bases isn't going to tell you much of anything, if it was even part of a gene or just regulatory sequences. For preserved DNA to be valuable, it would need to be a long strand of a few hundred bases at least. I don't think anything like that has been found.

However, these results certainly do give one reason to hope :)

[Andreas Sturm]

If you have enough 20mers, you could find sequence overlaps? But I guess you rather need 70mers so you get something you're able to work with...

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[Cathal Garvey]

Gotta disagree with you. Peptides are small, and small fragments of
eukaryotic proteins can *easily* show "significant" if not perfect
homology with proteins from drastically different families/clades.

As you say, a snowball has better chances than a string of peptides.

Mind you, DNA has better odds (though still unlikely), yet I was
boggled to view the hit TED video on reverse-engineering dinosaurs from
chickens. He described the methods they attempted to recover DNA from
bone, chiefly involving dissolution of the bone matrix..using HCl. A
fantastic way to rapidly degrade your paleo-DNA.

[Nathan McCorkle]

On Wed, Jul 17, 2013 at 2:14 PM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> Gotta disagree with you. Peptides are small, and small fragments of
> eukaryotic proteins can *easily* show "significant" if not perfect
> homology with proteins from drastically different families/clades.
>
> As you say, a snowball has better chances than a string of peptides.
>
> Mind you, DNA has better odds (though still unlikely), yet I was
> boggled to view the hit TED video on reverse-engineering dinosaurs from
> chickens. He described the methods they attempted to recover DNA from
> bone, chiefly involving dissolution of the bone matrix..using HCl. A
> fantastic way to rapidly degrade your paleo-DNA.

Hey c'mon, you know he was probably just hyping HCl because it sounds
cool and dangerous, right? Lot's of solutions use HCl, but none of the
buffer ions are glamorous or scary sounding.

On that note, DNA is much more stable than protein or RNA when it
comes to basic solutions...

[Cathal Garvey]

Well, buffers sure, but by the time you're degrading a fossil bone
matrix, you've probably dropped below pH 4, right? DNA's not so happy
down there, and paleo-DNA is likely already extremely fragile; nicks,
fractures, deaminations and other chemical modifications, you name it.

I wouldn't be surprised if we have zero success in this area using
traditional enzymatic methods, as it's possible the chemical nature of
any "preserved" DNA has changed as much as the bone does during
fossilisation. Will we have to resort to chemistry, mass-spec? Or
chimeric polymerases that aren't choosy about backbone structure?

I'm as interested as any genuine scientist in the DNA of dinosaurs,
even if I think this far-back-looking de-extinction hype is a load of
wasted talent.

[Anselm Levskaya]

DNA just isn't that stable on geologic time scales.  It's half life, even in the best of frozen permafrosty circumstances, is around 500 years.  Basically, once you get down to around 30bp fragments, coupled with the fact that you have very little source material or population diversity, reassembling metazoan genomes becomes statistically futile.

The record for recovering ancient DNA was recently broken with a 700,000 year-old horse bone sample.  I think this is getting very near the limit of what we're going to be able to recover.  Who knows, maybe we'll push out to a few million years in special circumstances, but getting out to 100Mya, especially considering the environmental dynamism of any chunk of earth on geological timescales, is a bit past the limits of credulity.

With regards to reviving recently extinct animals, I think people are being very delusional about the technological difficulty of the problems.  We're decades away from being able to synthesize and handle animal-size genomes.  The construction problems of DNA synthesis grow exponentially with the increase in molecule length.  Past 100kb DNA starts getting very physically delicate and hard to even transform into bacteria, and past 1Mb there are serious problems just packaging the DNA.  Dealing with the 100Mb size scale of animal and plant chromosomes will require an entire chain of technology just to be able to keep the molecules intact.  We'll probably have to engineer some sort of specialized eukaryotic cell to package and maintain incomplete chromosomes in a silent state.  We'll have to figure out worlds more metazoan cell biology to decipher how to build suitable surrogate cells to reboot extinct chromosomes, etc. etc.

It's so hard to do that I think it a poor use of resources.  It's much more interesting to make smaller changes in existing organisms to render them into more understandable versions (by adding input/output 'hooks') of their natural cousins than it is to simply recreate once-existing creatures.  

As a professional synbio person, the sad thing to me about synthetic biology so far is that all the big stunt projects are simply efforts to copy extant chromosomes/genomes rather than prove the immense, untapped potential of introducing new synthetic variety in reverse-engineering the logic of life.  Too much technique, not enough art. ;)

-A

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[A.J. DNA]

It depends on the protein, naturally. If you're looking at something as conserved as histones, then yes I'll grant you there's likely quite a bit of similarity between an animal and a fungus (my eukaryotic example contaminant from earlier). It would be very difficult for a 10 aa peptide to distinguish that. However, if you're looking at collagen, actin, or other structural proteins, which were also discovered in the T. rex and B. canadiensis fossils, something that varies much more from kingdom to kingdom, then you'll have better odds. Especially if you can find more than one peptide.

Peptides are much more likely to be unique in sequence than DNA due to the fact you have 20 possible choices for each component in the chain, whereas DNA only has 4. It's the same principle that allows song-matching apps to hear only ~20 sec of a song before IDing it. But like I said, it's tougher to rule out animal contamination. Maybe the fact that the peptides in question were buried inside the bone that would be inaccessible to multicellular organisms.

But there are statistical tests, and various algorithms to determine false discovery rates and so forth that give a little more robustness to the study. They also talk about finding DNA in the osteocytes, but are very guarded about whether its of dinosaurian origin. You don't have to take my word for it, you can read all about it in the paper (ain't science great?). Stay tuned.

[A.J. DNA]

I dislike the argument that "hard = waste of time". That's a slippery fucking slope.

But you're right, if I'm going to see a "dinosaur" in my lifetime that doesn't look like a bird, syn bio and reverse engineering a chicken is almost certainly how it's going to be done. But the result of that isn't going to be a "dinosaur" any more than the bird was. It's going to be the artist's/engineer's conception of what a "dinosaur" looks like, and thus will look cool and dinosaury but be inauthentic.

If you want a real DINOSAUR, you gotta go to the source. And yes, it will take a LONG time and a LOT of hard fucking work. But it's not like there will be nothing to show for it until the finish line is crossed and the first baby V. mongoliensis pops out of that egg. There are many, many discoveries hidden in the paleomolecular data waiting to be uncovered, perhaps something that changes how we see dinosaurs forever, like feathers. The stone has just been overturned. And I, for one, am happy to "waste my talent" to see what sort of cool stuff there is to find.

[Cathal Garvey]

The argument isn't "Hard = Waste of Time", the argument is "Hard with
little point = Waste of Time". That's pretty normal arithmatic for
deciding where to expend effort in every branch of human endeavor.

Not that you're prevented from doing so and expending your efforts and
talents towards reviving dinosaurs. Just don't get cross when we lament
what we perceive as waste. All that money and effort could be
preventing extant species from joining the "de-extinction" queue, after
all.

Whales/Tuna you can't eat. Pygmy tigers. Temperate-adapted rainforest
flora, to assist in north/south spread during rapid global warming.

That's where radical synbio can help prevent extinctions that will have
a real impact on the modern biosphere, instead of "reviving" species
that are probably no longer viable in our climate, and have no
ecological niche in which to naturalise.

If "de-extinction" is such a pernicious trend that you just have to do
it to look your buddies in the eyes again, then we have recently lost
plenty species that are far more relevant and valuable than Dinosaurs.
Or, if not recent, then within the timespans we're discussing as
realistic for DNA. What about that wonderful contraceptive plant the
Romans used to use, now extinct? De-extincting a plant is probably far
easier than an animal, after all, and we could totally use a
safe contraceptive prototype for synbio solutionism.

So, yea. Don't be offended, because it's not personal, but I do see the
zombie-species hype as being a waste of talent.

On Wed, 17 Jul 2013 18:33:09 -0700 (PDT)
"A.J. DNA" <immortal...@gmail.com> wrote:

> I dislike the argument that "hard = waste of time". That's a slippery
> fucking slope.
>
> But you're right, if I'm going to see a "dinosaur" in my lifetime
> that doesn't look like a bird, syn bio and reverse engineering a
> chicken is almost certainly how it's going to be done. But the result
> of that isn't going to be a "dinosaur" any more than the bird was.
> It's going to be the artist's/engineer's conception of what a
> "dinosaur" looks like, and thus will look cool and dinosaury but be
> inauthentic.
>
> If you want a real DINOSAUR, you gotta go to the source. And yes, it
> will take a LONG time and a LOT of hard fucking work. But it's not
> like there will be nothing to show for it until the finish line is

> crossed and the first baby *V. mongoliensis* pops out of that egg.

> > <nmz...@gmail.com<javascript:>

> > > wrote:
> >
> >> On Wed, Jul 17, 2013 at 2:14 PM, Cathal Garvey
> >> <cathal...@cathalgarvey.me <javascript:>> wrote:
> >> > Gotta disagree with you. Peptides are small, and small fragments
> >> > of eukaryotic proteins can *easily* show "significant" if not
> >> > perfect homology with proteins from drastically different
> >> > families/clades.
> >> >
> >> > As you say, a snowball has better chances than a string of
> >> > peptides.
> >> >
> >> > Mind you, DNA has better odds (though still unlikely), yet I was
> >> > boggled to view the hit TED video on reverse-engineering
> >> > dinosaurs from chickens. He described the methods they attempted
> >> > to recover DNA from bone, chiefly involving dissolution of the
> >> > bone matrix..using HCl. A fantastic way to rapidly degrade your
> >> > paleo-DNA.
> >>
> >>
> >> Hey c'mon, you know he was probably just hyping HCl because it
> >> sounds cool and dangerous, right? Lot's of solutions use HCl, but
> >> none of the buffer ions are glamorous or scary sounding.
> >>
> >> On that note, DNA is much more stable than protein or RNA when it
> >> comes to basic solutions...
> >>
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[A.J. DNA]

Not offended. Just passionate about dinosaurs :)

And you're focused on the destination, whereas my point is that it's the journey that makes it worth while. If you cannot see the point in expanding the scientific horizon, I would hesitate to call you a scientist. That's engineer talk :P Perhaps youre simply biased by the awesome and more realistic, tangible potential that synthetic biology has to offer. After all, who doesn't want to play god?

But I am, at heart, a dreamer, and a counterfeit dinosaur is no substitute for the dream.

Your other point about resources being better spent on preventing/mitigating the sixth mass extinction equally applies to just about every area of science I can think of, including cancer research. I'd happily go out of the job if it meant sparing the rest of the biosphere from the monsters that we are. Alas, we are such narcissistic fools.

But maybe someday. after we are all that remains (if we don't destroy ourselves first, I mean), we'll find a way to bring them all back. Wouldn't that be grand?

[Andreas Sturm]

>He described the methods they attempted to recover DNA from
bone, chiefly involving dissolution of the bone matrix..using HCl. A
fantastic way to rapidly degrade your paleo-DNA.

Ok, but how would it be possible to dissolve it? unsing just deionized water+ethanol which takes up the limestone? The ethanol for not dissolving DNA

>DNA just isn't that stable on geologic time scales.  It's half life, even in the best of frozen permafrosty circumstances, is around 500 years.

Wasn't the same thought for peptides? :D

>It's so hard to do that I think it a poor use of resources.

Yes, but you'll learn a lot of things that aply for other uses...

>  It's going to be the artist's/engineer's conception of what a "dinosaur" looks like, and thus will look cool and dinosaury but be inauthentic.

Did they have feathers? Very likely yes, but we'll know it for sure if we clone one :D


Is there a DNA Polymerase that doesn't need a primer? Or can one be engineereed?

Or is there a way to read the DNA without amplifying?

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[Mega]

I guess nature won't have created a DNA polymerase that doesn't need a primer, right?

So you would have to look at a RNA polymerase, applying various changes to the DNA polymerase and see which one may be the change that makes it dependent on primers...

To express it in bacteria, the new polymerase of course would need to be inhibited by a antibody (that falls of at >50°C) or something thelike. Because else, obviously, the bacterium would get millions of copies of its DNA and die...

[Cathal Garvey (Tablet)]

Actially, not a bad idea to use neutral or slightly acidic water in ethanol base. Rinse over bone freexing cold (calcium dissolves best in cold), feed into hot pipework to deposit calcium, cool and repeat. Cyclical dissolution/deposition of fossil elsewhere, leaving a reduced, more porous matrix.

Add amply chelators to improve carrying capacity at higher pH, capture stray nucleo-polymers on silica on way out prior to heat-deposition.

Would take ages though at neutral pH. Days, maybe weeks? Test it out on a lump of limestone! :)

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[Mega]

So it has to be done as cold as possible, so the DNA won't degrade... To sum up:

Dino-DNA extraction buffer stock solution:

20L nuclease free !! water
60L Ethanol
pH to 5.5 with HCl
*some* EDTA
other additives that block DNAses.

[Mega]

>Add amply chelators to improve carrying capacity at higher pH, capture stray nucleo-polymers on silica on way out prior to heat-deposition.

You wouldn't do this in one step, or did I get it wrong?

First, extract the limestone over a few weeks. The ethanol keeps the DNA solid/precipitated to the left over matrix (will there be a solid matrix left over?) or the DNA will sink to the ground

Then discard the supernatant, add water to dissolve th DNA.

[Mega]

One thing:
It was said that there is a maximum selflive for DNA.

But in the rock the DNA is captured in a dry state, protected from UV radiation.

So isn't it believable that the DNA survived to some degree?

[A.J. Devaux]

It's protected from UV, but not gamma, which permeates rock plenty over 65my, and causes DSBs. Big problem. I think the focus on DNA is not only unwarranted, but will cause some frustration as to feasibility. Like I said, you need long DNA stretches to get some actual info. But you can infer some structure and function with short polypeptides. We should try to work out how they worked at the cellular level before we can grow whole beasties. They skip that step in JP, I know. Movie science!

But I'm liking this protocol talk for demineralization. What I've read, they simply add 1 M EDTA in tris, pH 8, let it sit, spin down and repeat til there are no visible changes. But that was just for peptides. Is there a reason that wouldn't work for DNA? I'm no biochemist.

~A.J.

<typed with thumbs>

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[Cathal Garvey]

Entropy is the enemy. Even in the absence of any specific thing to
degrade it, DNA will gradually degrade due to factors such as latent
heat, isotopes forming the polymer undergoing spontaneous decay,
deep-penetrating cosmic radiation, and plenty of other factors.

As to the method I was suggesting, for super-cautious demineralisation I
suggested a cyclical system that re-uses the same buffer continuously,
but heats and cools the buffer in different stages to dump calcium and
resorb calcium, respectively.

In contrast to most minerals/salts, Calcium minerals/salts are generally
more soluble at lower temperatures, and less soluble at higher
temperatures. This is why scale builds up so quickly on kettle elements;
it's not that the carrying water all evaporates (although that
contributes), it's that the calcium in the cold water becomes insoluble
at higher temperatures.

You can drastically increase the calcium carrying capacity of your water
by lowering the pH, but too low and you start to destroy your DNA, too.

So, some undefined buffer consisting of a DNA precipitant like ethanol,
chelating agents to help protect DNA and improve solubility for calcium
and other minerals, and chilled to 4C, rinsed over the fossil. As it is
collected, it's passed over silica to bind any stray DNA that came
loose, and then heated to decalcify it so it can be re-used after cooling.

Technically, you don't have to cycle anything, you could just re-apply
fresh buffer regularly, but given that you want to keep the pH close to
neutral, it'll take a long time and waste a lot of buffer that way. A
cycle helps minimise waste and effort; set up, close the system, and
leave until the bone has degraded considerably. Then attempt DNA
extraction from the silica bed and the remaining fossil matrix.

This could, I should stress, all be complete crap.

[Andreas Sturm]

>but given that you want to keep the pH close to
neutral, it'll take a long time and waste a lot of buffer that way.

Surely you are true. But dinosaur DNA is worth billions of dollars, priceless. I would definitely sacrifice 10000 liters of buffer for getting some dino DNA (sequences).

Of course, the less needed, the better. Better for your pocket, better for the environment...

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[Nathan McCorkle]

On Jul 18, 2013 12:01 AM, "Andreas Sturm" <masters...@gmail.com> wrote:
>
> >He described the methods they attempted to recover DNA from
> bone, chiefly involving dissolution of the bone matrix..using HCl. A
> fantastic way to rapidly degrade your paleo-DNA.
>
> Ok, but how would it be possible to dissolve it? unsing just deionized water+ethanol which takes up the limestone? The ethanol for not dissolving DNA
>
>
>
> >DNA just isn't that stable on geologic time scales.  It's half life, even in the best of frozen permafrosty circumstances, is around 500 years.
>
> Wasn't the same thought for peptides? :D
>
>
>
>
> >It's so hard to do that I think it a poor use of resources.
>
> Yes, but you'll learn a lot of things that aply for other uses...
>
> >  It's going to be the artist's/engineer's conception of what a "dinosaur" looks like, and thus will look cool and dinosaury but be inauthentic.
>
> Did they have feathers? Very likely yes, but we'll know it for sure if we clone one :D
>
>
> Is there a DNA Polymerase that doesn't need a primer? Or can one be engineereed?

Nature already did that. It's called tdt or terminal deeoxynucleotide transferase,  or simply terminal transferase.

[Mega]

So that  terminal deeoxynucleotide transferase can take a single copy of DNA and amplify it? Sounds awesome...

Is this already used for sequencing ancient genomes?


How do you sequence it then, btw?



Let the terminal eoxynucleotyl transferase work for just a few seconds, so it makes a primer?

[Nathan McCorkle]

Sorry I meant it doesn't need a template! For something without a known primer, you simply ligate on a know sequence and use a primer for that

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