Journal of Applied Genetics 41(4), 2000, pp. 303-315
Adaptor-mediated amplification of minute amounts of severely fragmented ancient
Carsten M. PUSCH, Nikolaus BLIN, Martina BROGHAMMER, Graeme J. NICHOLSON, Michael SCHOLZ
Abstract: It has been repeatedly shown that high copy number mitochondrial DNA sequences can be recovered from ancient samples. A significant increase in the volume of information available to researchers will be observed when the amplification of nuclear DNA becomes commonplace and reproducible. To this end we established a modification of the Rapid Amplification of cDNA Ends (RACE) procedure normally used for the generation of cDNA ends from adaptor-ligated expressed sequence tag libraries. The modifications were designed to specifically address the problems associated with the highly damaged nucleic acids extracted from palaeontological specimens. For this study we used 6 human samples dating to 450 AD and ~6.500 BP that were refractory to reliable amplification of single copy loci by PCR. Racemate contents (ratio of D/L enantiomers) of aspartic acid, alanine, and leucine also indicated that no amplifiable DNA is present in 5 of the 6 samples. The proposed technique allowed us (i) to amplify four X-chromosomal loci from 5 human specimens, and (ii) to correct allelic drop-out phenomena at the amelogenin locus in one individual; thus showing that the threshold of 80 × 10-3 for D/Lasp as a borderline for the presence/absence of amplifiable aDNA requires reassessment. Reliability of the proposed technique (i.e. amplification of DNA sequences endogenous to the find) was validated by the application of "ancient RACE" (aRACE) to prehistoric animal samples.
Key words: adaptor ligation, ancient DNA, PCR, RACE, sex typing, short tandem
repeats, template improvement.
Correspondence: C.M. PUSCH, Molecular Genetics Laboratory, University Eye Hospital II, Auf der Morgenstelle 15, D-72076 Tubingen, Germany, e-mail: email@example.com