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Nucleic Acids Research, 2002, Vol. 30, No. 4 e16
© 2002 Oxford University Press

Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains

Giovanni Di Bernardo, Stefania Del Gaudio, Marcella Cammarota, Umberto Galderisi, Antonino Cascino* and Marilena Cipollaro

Department of Experimental Medicine and CRISCEB, 2nd University of Naples, Via Costantinopoli 16, 80138 Naples, Italy

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes ({varepsilon} globin, p53 and {gamma} interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to almost 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

* To whom correspondence should be addressed. Tel: +39 081 5665879; Fax: +39 081 5667547; Email: antonino.cascino{at}unina2.it +AF361529–AF361546, AF362488, AF427134, AF427135


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