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Author Topic:   Neanderthals and Cro-Magnon
Quetzal
Member (Idle past 5871 days)
Posts: 3228
Joined: 01-09-2002


Message 31 of 87 (42207)
06-06-2003 2:13 AM
Reply to: Message 30 by Mammuthus
06-05-2003 9:12 AM


Hi Mammuthus.
Can't the contamination problem apply to just about any "old" DNA experiment? I think you brought this up as your argument questioning the validity (during one of the interminable Borger discussions) of the Mungo Lake data. Is there some way to prevent contamination? How do you tell a "real" from a "contaminated" sequence? (In words of one syllable or less for all us non-molecular biology types... )

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Mammuthus
Member (Idle past 6474 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 32 of 87 (42210)
06-06-2003 4:21 AM
Reply to: Message 31 by Quetzal
06-06-2003 2:13 AM


Hi Quetzal,
The contamination problems (plural as there are many different problems) not only applies to old or ancient DNA but to modern samples as well. However, the low copy and degraded state of ancient DNA exacerbates the problem. Iwill try below to show why human or primate ancient DNA is particularly tricky relative to say work with mammoths.
Problems:
1. There is more DNA in a few cells of skin you shed than there probably is in most ancient human fossils. What this means is by the time you get the fossil from somebody who will allow you to whack it to pieces to extract DNA from it, lots of people have touched it with bare hands..not to mention those who did the excavation. If you cannot sample from an internal part of the sample i.e. taking a bone core then you will have a major contamination source. As Cooper in the Nature commentary states and has been demonstrated by others over the years, soaking in acid, bleach, UV light does not necessarily get rid of all contamination.
2. PCR primers that work for humans usually work for their relatives or near relatives. If I design PCR primers for mtDNA of Homo sapiens..they will work for Cro-Magnon as well. Contrast this to mammoths. I can design (and have frequently) primers that are specific for elephants. You can spike a reaction with human DNA and still never amplify it..so my Russian curator friends can touch the bones all they want and it won't cause me any problems.
3. Mungo Lake suffered from a different problem which plagues ancient DNA as well which are nuclear insertions of mtDNA or NUMTS. These are copies of mtDNA that have wandered into the nucleus and are now pseudogenes...there are hundreds if not thousands in the human genome...they often look very much like human mtDNA for example but often show differences from most bona fide mtDNA...they can be so divergent that they fall outside of the genetic variation of mtDNA for the human species i.e. like the neandertal type specimen sequence. That is why Krings et al. spent so much time and experimentation to support that the neanderal sequences was not an insert. The Mungo Lake sequence phylogenetically clustered with a known human mtDNA NUMT which is a big heads up that you have contamination...the most famous example is the claim that dinosaur DNA had been sequenced and it turned out to be a human nuclear insert sequence..doh!
With the above in mind...what do you do with ancient human samples? If the samples is contaminated your negative controls could still look good since your reagents are presumably not contaminated. You extract from a Cro-Magnon or a neandertal and get a sequence that looks like an average Joe mtDNA sequence. You send it to another lab to replicate the result (and since they will have the same contaminated sample) they may reproduce you contamination result...you can test multiple different samples and see if the results are consistent i.e. is the next Cro-Magnon sequence from another location similar or radically different? But if it comes back as a different common human mtDNA haplotype..what then? If it is completely different...which sequences is correct? Is one a contaminant? Are both?
Thus, you end up only accepting sequences that look very different from modern human mtDNA and that can be confirmed as non-nuclear in origin...and I think it is pretty pointless to do that much work if you cannot accept basically an entire array of data as valid because of contamination fears. Of course accepted neandertal sequences will be different from modern human since if a neandertal sequence with modern human sequences is found it will be dimissed as contamination!
Contamination, cross contamination, NUMTS all plague modern DNA studies as well...but a modern sample usually has so much DNA in it that you would really have to contaminate it pretty badly. Numts are a bigger problem which the scientific community mostly ignores rather than addresses...for my part, I have switched to nuclear DNA markers for mammoth analysis since my work with elephants showed they are so loaded with NUMTS that it is almost impossible to figure out what the bona fide mtDNA sequence is for the regions of the mitochondria that would interest me...of course, since so few mammoths yield nuclear DNA it has slowed my research down..but I rather deal with that then publish a completely screwed data set...and besides..its not like there is a big community of people working on mammoths...so my competition tends to be minimal compared to my more medically oriented work..so I can afford to be more plodding in my progress ...how is that for an excuse for being lazy

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Jor-el
Inactive Member


Message 33 of 87 (325791)
06-24-2006 7:07 PM


Bottleneck in human DNA Diversity
I know this an old topic but could you explain to me how this bottleneck 40000 yrs ago could relate to another such occurence 74000 yrs ago. The eruption of the supervolcano Toba in Sumatra, Indonesia, that is supposed to have been the cause of the last ice age according to some articles I've read.
It seems to me that these events have happened fairly often in human history. Although we haven't been hit that hard by anything major in the last 10000 yrs or so.
Also could you explain how one can compare DNA samples of hominids in these distant periods when DNA starts breaking down along with the tissue samples they are supposed to be taken from as soon as the being dies?
Logic would dictate that after so many millenia the DNA would be corrupted beyond salvage, although I may be working under a false premise when stating the above.
I'm still trying to learn the basics on this subject and it's tough without some basic explanations. Thanks for any input that you might be willng to give me.

We are the sum of all that is, and has been. We will be the sum of our choices.

Replies to this message:
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crashfrog
Member (Idle past 1466 days)
Posts: 19762
From: Silver Spring, MD
Joined: 03-20-2003


Message 34 of 87 (326084)
06-25-2006 11:05 AM
Reply to: Message 33 by Jor-el
06-24-2006 7:07 PM


Re: Bottleneck in human DNA Diversity
Logic would dictate that after so many millenia the DNA would be corrupted beyond salvage, although I may be working under a false premise when stating the above.
My guess, based on a few undergrad courses in genetics and my wife's research in insect molecular phylogenetics? It's not so much that DNA sequences become "corrupted" - nucleotide substitution isn't, to my knowledge, chemically likely in the environment of "decaying" DNA. It's that the DNA strands break into pieces.
But you can still PCR the pieces. Short strands of DNA are very stable. Assembling the whole strand from a random assortment of its pieces is a computationally intesnive, but not impossible, task. (Imagine if you had many copies of the same manuscript, each copy torn into tiny random pieces. For any two pieces that could be validly stuck together, there's probably a third piece from another copy that overlaps them, and tells you how they should be put together.)
So, not like computer data on a hard disk slowly degenerating into random 1's and 0's. More like the leaves of a book falling out of the spine and into random order.

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NosyNed
Member
Posts: 8996
From: Canada
Joined: 04-04-2003


Message 35 of 87 (326115)
06-25-2006 12:54 PM
Reply to: Message 34 by crashfrog
06-25-2006 11:05 AM


Re: Bottleneck in human DNA Diversity
This has nothing to do with the comparisons of DNA used to suspect a bottleneck.
The bottleneck is looked for by sequencing existing DNA and comparing the changes in various (I think non-coding) regions. The y chromosome and mitochondrial DNA are used to avoid the shuffling that others experience I think.

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Jor-el
Inactive Member


Message 36 of 87 (326510)
06-26-2006 3:36 PM
Reply to: Message 34 by crashfrog
06-25-2006 11:05 AM


Re: Bottleneck in human DNA Diversity
So what you're saying is that They can't recover a complete DNA sequence but only a multitude of part's of that same strand and then by using computer modelling they try to construct a DNA model of what the original strand looked like.
Since these small (stable) DNA strands are broken in various places they can use various parts to rebuild the whole with high degree of confidence that what they have is the same as the original.
Do I have this right or is there an error in my understanding?
I've also been reading up on the different types of studies with DNA.
The 1st being aDNA which is from what I've read the explanation which you have given me, if I understood correctly, and the 2nd is mtDNA which is the type of study that is used to study the Bottleneck in human DNA Diversity which I referred to in my post.
mtDNA, from what I've read is apparently the study most used due to its' reliability. Is there a site which explains the use of these different tests and their purposes that is not so technical that I wouldn't understand a word? I've been looking but can't seem to find anything that is directed toward instructing someone in laymans terms.

We are the sum of all that is, and has been. We will be the sum of our choices.

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randman 
Suspended Member (Idle past 4898 days)
Posts: 6367
Joined: 05-26-2005


Message 37 of 87 (326521)
06-26-2006 4:41 PM
Reply to: Message 3 by Mammuthus
05-19-2003 10:47 AM


Hmmm.....
My problem with these types of studies is I feel they are a bit rigged from outset. If one found a neanderal specimen that gave a sequence like that of the Cro-magnon, it would likely be dismissed as a lab contamination.
That statement doesn't exactly inspire confidence in the objectivity of the process.
Edit to add that you answered the "why" question earlier. It seems to me then that drawing conclusions from DNA studies of Neanderthals is a severely flawed process.
Edited by randman, : No reason given.

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crashfrog
Member (Idle past 1466 days)
Posts: 19762
From: Silver Spring, MD
Joined: 03-20-2003


Message 38 of 87 (326681)
06-26-2006 10:56 PM
Reply to: Message 36 by Jor-el
06-26-2006 3:36 PM


Re: Bottleneck in human DNA Diversity
So what you're saying is that They can't recover a complete DNA sequence but only a multitude of part's of that same strand and then by using computer modelling they try to construct a DNA model of what the original strand looked like.
That's my guess. It's the exact same technique, by the way, that is used to sequence the contemporary human genome. It turns out that you basically have to cleave genomic DNA into small pieces anyway, just to be able to sequence it sooner than a few decades.
mtDNA, from what I've read is apparently the study most used due to its' reliability. Is there a site which explains the use of these different tests and their purposes that is not so technical that I wouldn't understand a word? I've been looking but can't seem to find anything that is directed toward instructing someone in laymans terms.
The tests are usually always the same; PCR-RFLP or other kinds of tests. Basically, very simple ways to examine a single gene across multiple individuals and establish a phylogenetic tree of their relationships and shared ancestors.
I've been looking but can't seem to find anything that is directed toward instructing someone in laymans terms.
Offhand, I don't know. Maybe your best bet is to open a thread to discuss one of the really technical explanation, and ask the biologists here questions that will help it make sense to you.

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RAZD
Member (Idle past 1404 days)
Posts: 20714
From: the other end of the sidewalk
Joined: 03-14-2004


Message 39 of 87 (326694)
06-26-2006 11:51 PM
Reply to: Message 1 by Mammuthus
05-16-2003 8:56 AM


Hey Mammuthus. Long time no see.
Do you know of any studies that compare Y chromosomes? It seems to me that only neander moms have been eliminated with mtDNA tests, leaving the possibility of neander dad DNA being involved.
If the hybrids were only fertile in one combination and not the other (as happens in mules?), this could still leave interbreeding AND the mtDNA divisions as possibilities.

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Mammuthus
Member (Idle past 6474 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 40 of 87 (326720)
06-27-2006 3:36 AM
Reply to: Message 37 by randman
06-26-2006 4:41 PM


Re: Hmmm.....
Perhaps, however, the objectors are memmbers of the one group that has the most to lose which is the group that sequenced the first neandertal and want it to be different. However, the objectivity in the process comes from the dozens of labs that have attempted to pick apart the neandertal results..and thus far have been unable to..in fact, every subsequenct neandertal sequenced has reinforced the conclusions of the first study. The point will soon be moot as using a new pyro sequencing method that was used to sequences about 1% of the mammoth genome, the neandertal nuclear genome is being queued up for sequencing. If they get enough sequence, this will provide the kind of data necessary to determine if the "different species" status neandertals currently enjoy based on mtDNA is supported by nuclear markers. Or perhaps nuclear genes will support neas as a different species but that there was some admixture. Remains to be seen.
quote:
It seems to me then that drawing conclusions from DNA studies of Neanderthals is a severely flawed process.
Going in to the DNA based projects, nobody had any idea what the results would be..the results have been scritinized by dozens of groups and has held up...more groups are scritinizing the results still. The Cro-Magnon results have not been held to this kind of scritiny yet.
Science is not like religion where when something is wrong in religion, it is covered up. There is always somebody out there in science trying to knock down your conclusions or expand on your findings...or beat you to it. Not the mindless orthodoxy demanded by conservative relgions.
There is nothing flawed about the conclusions drawn. From the 7 or so neandertals sequenced from very different localities thus far, there is absolutely no evidence of either being human or having mixed with human populations. However, the conclusions are tentative for the reason I gave that it is not possible to distinguish a contaminant from endogenous DNA if the sequence were to be more human like...thus far this has not occurred for neandertals but is exactly what happened with Cro-Magnon. Whole genome (or large scale)sequencing will resolve the issue....again, science moves forward...you should try it sometime.
Edited by Mammuthus, : No reason given.

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Mammuthus
Member (Idle past 6474 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 41 of 87 (326721)
06-27-2006 3:43 AM
Reply to: Message 39 by RAZD
06-26-2006 11:51 PM


As I mentioned in my response to randman, the nuclear genome of the neandertal type specimen is being cued up for sequencing. This will provide the kind of information that is needed to address the question of admixture. What you describe is seen in the two species of African elephants, the forest and savana elephants. It is called cytonuclear disocciation. What happens is that the much larger savana bulls outcompete the smaller forest bulls in areas where the two species meet. The resulting hybrids are half forest half savana at nuclear loci but pure forest for mtDNA. What has happened as a result is that you have some savana elephants that have almost completely savana nuclear genomes (because of backcrossing of to savana males) but have forest mtDNA.
It may turn out that something like this exists between humans and neandertals i.e. there may be a few human individuals with nea like mtDNA but pure human nuclear and vice versa...or it could be that there was zero admixture.

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Jor-el
Inactive Member


Message 42 of 87 (326890)
06-27-2006 4:00 PM
Reply to: Message 41 by Mammuthus
06-27-2006 3:43 AM


What would, in your opinion, be the maximum age limit for a specimen, before DNA in these types of studies becomes unusable due to degradation of one type or another?
"The biggest problem is that DNA begins to degrade from the moment of death as water, oxygen, and microbes attack it."
Patricia Kahn and Ann Gibbons
Copyright © 1997 by the American Association for the Advancement of Science.
ANTHROPOLOGY: DNA From an Extinct Human

We are the sum of all that is, and has been. We will be the sum of our choices.

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Mammuthus
Member (Idle past 6474 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 43 of 87 (327054)
06-28-2006 3:53 AM
Reply to: Message 42 by Jor-el
06-27-2006 4:00 PM


Hi Jor-El
Regularly reported and verified extraction and characterization of DNA from samples in the 40-50,000 year old range are abundant i.e. woolly mammoths. I think about the oldest reproduced sequences came from 80 Ky bears. The limit will probably be around 100 K years unless a find is made in an unusual preserving context. Note, the one such context examined, insects encased in amber, did not pan out as none of the studies were reproducible.
Most ancient DNA studies focus on samples in the age range from early Holocene to late Pleistocene (anywhere from a few thousand years to about 30,000 years ago).

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ramoss
Member (Idle past 611 days)
Posts: 3228
Joined: 08-11-2004


Message 44 of 87 (327116)
06-28-2006 10:16 AM
Reply to: Message 43 by Mammuthus
06-28-2006 3:53 AM


Limit of 100K years, huh? I guess no jurrassic parks will be made.

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Mammuthus
Member (Idle past 6474 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 45 of 87 (327122)
06-28-2006 10:23 AM
Reply to: Message 44 by ramoss
06-28-2006 10:16 AM


I doubt there will even be Pleistocene or early Holocene Parks made. Even if one were able to sequence the entire genome of say a mammoth, how would you actually make a mammoth? So far cloning has involved nuclear transfer...not just blasting DNA into a denucleated cell...maybe someday down the road..but not anytime soon.
Jurassic DNA probably completely degraded in the Jurassic regardless of how well preserved (morphologically) insects in amber may be.

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