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

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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.

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