To: Jeff Doles 940429 19:%:00 Subject: Re: dna dating = Quoting Jeff Doles to All = JD SCI

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.