Amino acid racemization and the preservation of ancient DNA

Science

Volumn 272, Issue 5263, 1996, Pages 864-866

  Poinar, Hendrik N ^a   Höss, Matthias ^a,c   Bada, Jeffrey L ^b   Pääbo, Svante ^a  

^a UNIVERSITY OF MUNICH   (Germany)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c IMPERIAL CANCER RESEARCH FUND   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

ALANINE; AMBER; ASPARTIC ACID; DNA; LEUCINE;

ARTICLE; DNA DETERMINATION; DNA SEQUENCE; HUMAN; INSECT; NONHUMAN; PRIORITY JOURNAL; RACEMIC MIXTURE;

HEXAPODA; INSECTA;

AMINO ACID; DNA; FOSSILIZATION; PRESERVATION; RACEMIZATION;

EID: 0029663543     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5263.864     Document Type: Article

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29. External surfaces of bone sections (∼1 mm) were removed, and samples were ground under liquid nitrogen in a Freezer/Mill 6700 bone grinder (Spex Industries, Edison, NJ). Then 0.01 to 0.5 g were hydrolyzed in doubly distilled 6 N HCI for 24 hours at 100°C. Glassware was cleaned by immersion in 10 M HCl for 2 weeks, then rinsed in doubly distilled water and baked at 250°C for 1 week. Soft tissue samples were briefly rinsed in 0.01 N HCl and hydrolyzed as above. Subsequently, samples were dried under vacuum over NaOH, Bone samples were redissolved in doubly distilled water and desalted with a cation exchanger (50W-X8) (Bio-Rad) as in J. L. Bada, Earth Planet. Sci. Lett. 15, 223 (1972). Soft tissue samples were dissolved directly in 0.4 M sodium borate. Amino acids were derivatized with O-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC) and analyzed by high-pressure liquid chromatography (HPLC) (Gilson, Middleton, WI) with fluorescent detection as described in M. Zaho and J. L. Bada, J. Chromatogr. A 690, 55 (1995). Mock samples were analyzed in parallel with each series of samples, and background amino acid concentrations were subtracted from sample values. For quantitation and accurate determination of enantiomeric ratios, a standard containing a racemic mixture of the selected amino acids was analyzed on the same day as the samples. To determine the extent of racemization caused by the experimental procedure, we analyzed five bovine serum albumin (Pharmacia) samples; they were found to have a D/L ratio for Asp of 0.034 ± 0.0035. Variation due to sample processing was tested by the analysis of 10 samples from the same bone, yielding a D/L ratio of 0.056 ± 0.0036. We investigated the relative fluorescence of OPA-derivatized D and L forms of Asp, Ala, and Leu by analyzing samples with D/L ratios of 1. The observed ratios (0.867, 0.85, and 0.80, respectively) were used to adjust values determined for the samples.
30. External surfaces of bone sections (∼1 mm) were removed, and samples were ground under liquid nitrogen in a Freezer/Mill 6700 bone grinder (Spex Industries, Edison, NJ). Then 0.01 to 0.5 g were hydrolyzed in doubly distilled 6 N HCI for 24 hours at 100°C. Glassware was cleaned by immersion in 10 M HCl for 2 weeks, then rinsed in doubly distilled water and baked at 250°C for 1 week. Soft tissue samples were briefly rinsed in 0.01 N HCl and hydrolyzed as above. Subsequently, samples were dried under vacuum over NaOH, Bone samples were redissolved in doubly distilled water and desalted with a cation exchanger (50W-X8) (Bio-Rad) as in J. L. Bada, Earth Planet. Sci. Lett. 15, 223 (1972). Soft tissue samples were dissolved directly in 0.4 M sodium borate. Amino acids were derivatized with O-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC) and analyzed by high-pressure liquid chromatography (HPLC) (Gilson, Middleton, WI) with fluorescent detection as described in M. Zaho and J. L. Bada, J. Chromatogr. A 690, 55 (1995). Mock samples were analyzed in parallel with each series of samples, and background amino acid concentrations were subtracted from sample values. For quantitation and accurate determination of enantiomeric ratios, a standard containing a racemic mixture of the selected amino acids was analyzed on the same day as the samples. To determine the extent of racemization caused by the experimental procedure, we analyzed five bovine serum albumin (Pharmacia) samples; they were found to have a D/L ratio for Asp of 0.034 ± 0.0035. Variation due to sample processing was tested by the analysis of 10 samples from the same bone, yielding a D/L ratio of 0.056 ± 0.0036. We investigated the relative fluorescence of OPA-derivatized D and L forms of Asp, Ala, and Leu by analyzing samples with D/L ratios of 1. The observed ratios (0.867, 0.85, and 0.80, respectively) were used to adjust values determined for the samples.
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32. For example, the total amounts of the six amino acids analyzed [Asp, serine (Ser), Ala, Gly, Leu, and valine (Val)] do not correlate with the retrieval of DNAsequences (H. N. Poinar, M. Höss, J. L. Bada, S. Pääbo, data not shown). Furthermore, a ratio of Gly to Asp of 5.5 or larger can be used as a rough estimate of the preservation of collagen in bone [H. Elster, E. Gil-Av, S. Weiner, J. Archeol. Sci. 18, 605 (1991)]. However, DNA could be extracted both from bones in which collagen was preserved according to this criterion and from some in which it was not.
33. Only minute amounts of amino acids were present in these samples, and these values were not significantly different from those in the surrounding sediment. As a result, the D/L ratios for Asp could not be determined in most cases. The presence of bacterial amino acid decomposition products, for example, β-alanine and γ-amino-n-butyric acid, in these specimens also suggests bacterial contamination (J. L. Bada, unpublished observations).
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36. X. S. Wang, H. N. Poinar, G. O. Poinar Jr., J. L. Bada, in Amber, Resinite, and Fossil Resin, K. B. Anderson and J. C. Crelling, Eds. (American Chemical Society, Washington, DC, 1995), pp. 255-262.
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39. The Utah and Montana dinosaur samples were too small to allow for the removal of surface material. Instead, they were rinsed several times with 0.01 N HCI and then with doubly distilled water before hydrolysis. The remaining dinosaur bones were processed like the other bones (12). Clarkia sediments containing fossil leaves were opened in a hood, photographed, and rinsed with 0.01 N HCl. Leaves were then scraped off into sterile vials, hydrolyzed, desalted, and analyzed by HPLC (12). Surrounding sediments were similarly analyzed. Amber samples were processed as in (11).
40. We thank V. Börner, A. Cooper, O. Handt, M. Krings, G. O. Poinar, J. Rea, A. Sajantila, S. Weiner, S. Woodward, H. Zischler, and others for help, and J. E. Cadle, A. Currant, A. v.d. Driesch, U. Joger, H. G. McDonald, M. C. McKenna, C. E. Ray, C. A. Shaw, A. J. Sutcliffe, H. P. Lterpmann, M. K. Vereschagin, and M. Hanczyk for samples. This research was supported by the Deutsche Forschungsgemeinschaft (S.P.) and the NASA Specialized Center for Research and Training in Exobiology (J.L.B.).