From: Patrick O'Neil
To: Jeff Doles 94-04-29 19:%:00
Subject: Re: dna dating
-=> Quoting Jeff Doles to All <=-
JD> SCIENCE SPOT
JD> DNA dating:
JD> Fascinating evidence that the fossils are young
JD> Carl Wieland, M.B., B.S.
JD> ------------------------
JD> DNA, the complex molecule of heredity, can he observed in the
JD> lahoratory to hydrolyse (break down) by itself. We have already
JD> commented in Cleation magazine on the discovery of DNA in magnolia
JD> leaf fossils which are supposed to be around 20 million years old
JD> according to evolutionary assumptions, and suggested that this
JD> seemed rather unbelievable for a complicated molecule which
JD> progressively disintegrates all hy itself (Vol. 13 No. 2, pp. 22-23).
JD> Now Brian Sykes in the prestigious journal "Nature" clearly states
JD> that the rate at which DNA breaks down in the laboratory is such
JD> that after 10,000 years no DNA should be left. Writing ahout the
JD> magnolia leaf fossils (and others in the same 'ancient' layer found
JD> to also have DNA, including oak, cypress and tulip tree fossils) he
JD> says:
JD> 'This means these compression fossils defy the prediction, from in
JD> vitro estimates of the rate of spontaneous hydrolysis, that no DNA
JD> would remain intact much beyond 10,000 years. What a good job not
JD> every body knew that, grant reviewers included."
OK, I have here an article from _Nature_, vol 362, 22 April
1993, by Tomas Lindahl entitled, "Instability and decay of the
primary structure of DNA."
A few quick quotes to keep it short:
"For single-stranded DNA in solution, the half-life of an individual
cytosine residue is about 200 years at 37 degrees C and at pH 7.4."
--now of course, this is single-stranded DNA and refers to cytosine
as an example-it degrades quicker than the others.
"In contrast to to depurination, the double helix structure affords
very good protection against hydrolytic cytosine deamination, and this
reaction occurs at only 0.5-0.7% of the rate of single-stranded DNA, that
is, with a half-life of 30,000 yrs for each cytosine residue."
--as with radioactive dating, the half-life doesn't mean that after
60,000 yrs there wont be any cytosine residues. It means that after
30,000 yrs, half the cytosine will be degraded, after another 30,000
yrs half of the remaining half will be degraded, and so on.
--to directly address the ancient DNA question the following quotes:
"Ancient DNA
Recovery of DNA fragments from extinct animals or plants in museum
collections or from archaeological excavations can permit direct
comparison with related contemporary material by DNA sequencing. This
approach offers a valuable complement to taxonomic studies. Short
mitochondrial DNA sequences from the extinct quagga have been cloned and
compared with homologous sequences from zebra and other related animals.
Similarly, 2,400-year-old DNA fragments from an Egyptian mummy have been
cloned and characterized. The advent of the polymerase chain reaction
(PCR) has greatly aided such investigations because minute amounts of
DNA fragments can be selectively amplified. Thus DNA samples from
archaeological bones up to 5,000 year old have been recovered and
analysed. A problem with the hypersensitive PCR technique, as
encountered by most newcomers to the method, is that trace amounts
of contaminating DNA accidently derived from laboratory glassware, or
even the experimenter, readily produce false-positive results. This is
a particularly difficult problem in work with ancient DNA, which is
often highly degraded or possibly non-existent in available specimens
and the occasional dramatic 'success' in this area should be viewed with
skepticism.
From the data on hydrolytic and oxidative decomposition reviewed above,
it can be predicted that deprived of repair mechanisms provided by the
living cells, fully hydrated DNA is spontaneously degraded to short
fragments over a time period of several thousand years at moderate
temperatures. The most important route of decay for hydrated DNA is
depurination. A 5-10 fold reduction in the rate of this process can be
achieved at very high ionic strength. Furthermore, absorption of DNA
to hydroxyapatite results in a twofold decrease in the rate of
depurination; this could slightly improve the chances of recovery of
useful DNA sequences from old bones. Thus, in connection with favorable
preservation conditions, it seems feasible that useful DNA sequences
**tens of thousands of years old** ((my emphasis)) could be recovered,
particularly if the fossil has been retained at low temperature.
Preliminary findings have been made on the identification of short
mitochondrial DNA sequences from 40,000 yr old mammoth tissue. The data
on bacterial spores would suggest that further increased stabilization
of the DNA in fossils would be achieved by partial dehydration, and by
exclusion of oxygen. Note in this regard that DNA in air-dried tissues
remains partially hydrated and is still susceptible to decay. Because
some water molecules in the grooves of the DNA double helix are
structurally essential, 'dry DNA'obtained by storing DNA fibers over
the efficient drying agent phosphorus pentoxide does not retain a
double helical conformationn, making bases more vulnerable to damage;
such DNA is extremely hygroscopic and is rapidly rehydrated on exposure
to air.
Recently, much excitment has been generated by claims of PCR
amplification of DNA tens of millions of years old. The most intriguing
of these reports concerns DNA from a 25-30 million yr old termite
preserved in amber. The authors tried with commendable care to avoid
contamination artefacts that have marred much of the work in this novel
area of research. Minute amounts of highly degraded DNA were extracted
from the preserved termite under sterile conditions in a laboratory
where previous work on insect DNA had not been done. A number of PCR
control PCR amplifications were done to exclude any high-molecular
mass DNA from the analysis, because such DNA would probably be of recent
origin. In spit of these precautions, traces of several types of insect
DNA sequences were recovered from the fossil material, which were
'dipteran in general and drosophilid in particular.' The most highly
degraded DNA ( <250 base pairs) contained some termite-like sequences
as deduced from analysis of mitochondrial and nuclear ribosomal DNA;
these were somewhat arbitrarily assigned as being representative of the
fossil, whereas the other insects were deemed to be contaminants. It
will now be important to assess the reproducibility of such findings.
Amber preservation appears to provide a uniquely advantageous way of
retaining ancient DNA sequences, because the DNA is largely dehydrated,
partly protected form atmospheric oxygen, and not exposed to microbial
contamination."
--I will now quote, briefly, from an article in _Nature_, vol 363,
10 June 1993, entitled, "Amplification and sequencing of DNA from a
120-135 million-year-old-weevil," by Raul Cano, Hendrik Poinar,
Norman Pieniazek, Aftim Acra, & George Poinar:
"DNA has been successfully isolated from both fossilized plant and
animal tissues. The oldest material, dated as 25-40 million years old
(Tertiary), was obtained from amber-entombed bees and termites. Tissues
from both these insects yielded DNA of good quality, which could be
amplified by the polymerase chain reaction (PCR) and subsequently
sequenced, including the genes encoding 18S ribosomal RNA and 16S rRNA.
We report here the extraction of DNA from a 120-135 million yr old
weevil (Nemonychidae, Coleoptra) found in Lebanese amber, PCR
amplification of segments of the 18S rRNA gene and the internal
transcribed spacer, and the corresponding nucleotide sequences of
their 315- and 226-base-pair fragments, respectively. These sequences
were used for preliminary phylogenetic analysis of the nemoychid's
sequence with three extant coleopterans: Lecontellus pinicola
(Nemonychidae), Hypera brunneipennis (Curculionidae) and the mealworm
Tenebrio molitor (Tenebrionidae), and two extant dipterans: the
fruitfly Drosophila melanogastor (Drosophilidae) and mosquito
Aedes albopictus (Culicidae) for the purpose of ascertaining the
origin of the extracted and amplified DNA. The results revealed that
the PCR-amplified material is that of the extinct nemonychid weevil.
This represents the oldest fossil DNA ever extracted and sequenced,
extending by 80 million yrs the age of any previously reported DNA."
"These results demonstrate the successful extraction and amplification
of DNA from a 120-135 Myr-old amber fossilized insect. We speculate
that on the basis of our investigation that the majority of animal
remains in amber have preserved DNA taht can be extracted and sutdied,
thus making amber a treasure chest for molecular palaeontologists."
Since these articles, many other extractions have been accomplished
and contamination controlled for.