Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.
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 (epsilon 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 alpha(lambda)mu(omicron)sigma(tau) 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.
Di Bernardo
Giovanni
G
Department of Experimental Medicine and CRISCEB, 2nd University of Naples, Via Costantinopoli 16, 80138 Naples, Italy.
Del Gaudio
Stefania
S
Cammarota
Marcella
M
Galderisi
Umberto
U
Cascino
Antonino
A
Cipollaro
Marilena
M
D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000737:chemistry; D002467:Cell Nucleus / Q000235:genetics; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D011088:DNA Ligases / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry; D004260:DNA Repair; D018397:Equidae / Q000235:genetics; D049690:History, Ancient; D007558:Italy; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D010163:Paleontology; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment
D004247:DNA; D004256:DNA Polymerase I; D011088:DNA Ligases