Amplification and sequencing of DNA from a 120–135-million-year-old weevil

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Abstract
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-year-old weevil (Nemonychidae, Coleoptera) 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 nemonychid'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 melanogaster (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 years the age of any previously reported DNA.