Just adding to the palaeo-molecularological confusion (mine).

[Jacques Cinq-Mars]

All,

Does the following (see, for example, the "red" sentence) imply that all the work carried out so far on identifying and comparing palaeo-DNA sequences may be flawed in some ways? Is it a "back to the drawing board" question?

Jacques Cinq-Mars

Pusch, Carsten M.  and Lutz Bachmann. 2004. Spiking of Contemporary Human Template DNA with Ancient DNA Extracts Induces Mutations Under PCR and Generates Nonauthentic Mitochondrial Sequences. Mol. Biol. Evol. 21(5):957-964.

Abstract:

Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.

Key Words: ancient DNA extracts • mitochondrial DNA • mutation • PCR errors • PCR inhibition

Copyright © 2004 by the Society for Molecular Biology and Evolution.

[caldararo]

Dear Jacques:

    I do not think that this alone with do it, but together with the work by Gill Bejerano and David Haussler published in Science Express last week and the article by Gillooly, Allen et al., "Metabolic rate calibrates the molecular clock: reconciling molecular and fossil estimates of evolutionary divergence," (2004) should.  Gillooly, et al., examined the observation that rates of molecular evolution vary widely within and among lineages to find if there was one molecular clock mechanism.  They found that there is a general molecular clock but that it "ticks" at a constant substitution rate per unit mass-specific metabolic energy rather than per unit time.  This is a return to Morris Goodman's original perspective and Guthrie and I suggested the problem in 1998.  On the other hand, this is based on the assumption that neutral theory applies which has been thrown into question by the work by Bejerano and Haussler.  They found that a considerable number of intron sequences are exactly the same  across genera indicating that vast portions of the genome of most animal life is under some constraint from random change.  Pusch and Bachmann's work verifies Gabow and my findings in 2000 in Ancient Biomolecules.  Our papers are available online at http://eres.sfsu.edu/coursepage.asp?cid=1508.

Niccolo Caldararo, Ph.D.
Dept. of Anthropology
San Francisco State University

[Jacques Cinq-Mars]

Dear Niccolo,

Thanks for your response/clarification. It does help -- let's say, a little bit -- and, unless I am mistaken, my recent post, re: "Back to Darwinian Selection" '!', should make me realize that I shouldn't feel too guilty about being confused.

Jacques

PS Note that the URL you provided us with is a cul-de-sac in that a password is required in order to access the papers you referred to.

[caldararo]

Dear Jacques:

   Sorry, the password is "bark" which refers to how much like dog mtDNA we really are as well as varied phenotypically and interfertile.  By the way, now with the molecular DNA in "disarray", perhaps we can get back to talking about the Neandertal stage of human evolution with Kabwe and others as regional variants along the way to H.s.s.?  Stringer and Day's classification guidelines for early H.s.s are again open for debate as Woloff demonstrated years ago there lack of logic.

Niccolo Caldararo, Ph.D.
Dept. of Anthropology
SFSU

[Jacques Cinq-Mars]

Dear Jacques:

   Sorry, the password is "bark" which refers to how much like dog mtDNA we really are as well as varied phenotypically and interfertile.  By the way, now with the molecular DNA in "disarray", perhaps we can get back to talking about the Neandertal stage of human evolution with Kabwe and others as regional variants along the way to H.s.s.?  Stringer and Day's classification guidelines for early H.s.s are again open for debate as Woloff demonstrated years ago there lack of logic.

Niccolo Caldararo, Ph.D.
Dept. of Anthropology
SFSU

Dear Niccolo,

I suppose I should have done this sooner! Anyway, thanks for the reassuring words and for passing on the "password". It does work but, unfortunately (at least for me), does not provide access to the paper(s) you mentioned in your earlier post, but only to papers for your ANTH631 course.

Jacques

[Dale Hoogeveen]

Hi Jacques,

http://eres.sfsu.edu/courseindex.asp

Then select Anthropology in the course and Caldararo in the Instructor field.  That should list them both and it looks to me like the same password works on both of them.

Or go directly:

http://eres.sfsu.edu/coursepage.asp?cid=1459


That should get you to ANTH100.
The Caldararo/Gabow pdfs on mtDNA and Caldararo/Guthrie on Y chromosome are definitely worth the effort.

Dutch

[Jacques Cinq-Mars]

Hi Jacques,

http://eres.sfsu.edu/courseindex.asp

Then select Anthropology in the course and Caldararo in the Instructor field.  That should list them both and it looks to me like the same password works on both of them.

Or go directly:

http://eres.sfsu.edu/coursepage.asp?cid=1459


That should get you to ANTH100.
The Caldararo/Gabow pdfs on mtDNA and Caldararo/Guthrie on Y chromosome are definitely worth the effort.

Dutch

Dale,

Thanks for the electronic guidance. I followed your instructions and got the papers.

Now, what's next? Being that I am definitely more comfortable with bones, stones, geoarchaeology, etc., I do not intend to loose too much sleep over what I take to be an apparently increasing series (inner and outer) of contradictions  in the "molecular" palaeoanthropological discourse, but, nonetheless, it would be nice if someone, somewhere, were to come up with (at the very least) a half decent, logical calibration/harmonization of the various sorts/levels of evidence that are supposed to help us elucidate various aspects of prehistoric human biogeography.

Jacques