Selective amplification of a mammoth mitochondrial cytochrome b fragment using an elephant specific primer

• Published in: Current genetics 1995-04, Vol.27 (5), p.486-487
• Main Authors: Hauf, J, Baur, A, Chalwatzis, N, Zimmermann, F.K, Joger, U, Lazarev, P.A. (Technische Hochschule Darmstadt (Germany). Inst. fuer Mikrobiologie)
• Format: Article
• Language: English
• Subjects: ADN; Animals; Base Sequence; Cattle; CITOCROMO B; CYTOCHROME B; Cytochrome b Group - genetics; DNA; DNA Primers - genetics; ELEFANTE; ELEPHANT; ELEPHANTS; Elephants - classification; Elephants - genetics; Elephas maximus; EVOLUCION; EVOLUTION; Humans; Loxodonta africana; Mammuthus primigenius; MITOCHONDRIA; Mitochondria - enzymology; MITOCHONDRIE; MITOCONDRIA; Molecular Sequence Data; Paleontology; POLIMERIZACION; Polymerase Chain Reaction; POLYMERISATION; POLYMERIZATION; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/BF00311220

The Woolly Mammoth (Mammuthus primigenius) is considered to be a descendant of Mammuthus (Archidiscodon) meridionalis which was distributed from Southern Europe to India in the late Pliocene and the early Pleistocene. This evolutionary line may have originated in Africa 5 million years ago. The question is whether it is more closely related to the African, Loxodonta africana, or the Indian, Elephas maximus, elephant. All three genera are derived from the Pliocene Primelephas. Hoss et al. using PCR have amplified and sequenced 93 bp of the 16 RNA gene of four different mammoth carcasses from different locations in Siberia. The four mammoth sequences differed between each other and also from those of the Indian and African elephants. Hagelberg et al. amplified 283 bp from the mitochondrial cytochrome b gene of two mammoth carcasses from two locations in Siberia. These sequences differed at five positions and both different from those of the Indian and African elephants. DNA has a rather limited stability and thus amplification of ancient or even fossil DNA is fraught with considerable uncertainties caused by contamination with recent DNA. Our previous attempts to amplify mammoth DNA from nuclear 18 RNA and mitochondrial cytochrome b genes using "standard" primers yielded only contaminating Aureobasidium pullulans and human gene sequences respectively. Therefore, we tried to identify elephantid-specific base sequence motives in the cytochrome b gene in order to design primers which would exclude the amplification of human sequences. Data-bank comparisons showed that in the published information on the African elephant, L. africana (EMBL accession number X56285), the sequence AAG at positions 515-517 differed from the human GTC sequence (EMBL accession number M58503) but not from those of Bos taurus (EMBL accession number V00654), Mus musculus (EMBL accession number V00711), and Didelphis virginiana, (EMBL accession number Z29573). We designed a forward primer starting with position 493, and including position 517, 5'TGAGG(A/G)GGCTT(T/C)TCAGT(A/C)GACAAAG 3', and a reverse primer covering positions 623 through 601, 5'GG(A/G)TTGTT(T/G)GA(G/T)CCTGTTTCGTG 3'. All numbers refer to the complete cytochrome b gene of L. africana. We extracted DNA by the phenol-chloroform method from 10 g of a skin sample of a mammoth carcass found in 1992 in the banks of the Shandrin river, a tributary of the Kolyma, North Yakutia. DNA was purified over a Qiagen column to yield 40 mu