PREAMPLIFICATION PROCEDURE IN ANCIENT DNA SAMPLES
Stefania Del Gaudio1, Alessandra Cirillo1, Giovanni Di Bernardo1, Umberto Galderisi1, Theodoros
Pitsios2, Marilena Cipollaro1
1
Department of Experimental Medicine, Section of Biotechnology and Molecular Biology "A. Cascino",
Second University of Naples, Naples, Italy; 2 Museum of Anthropology, University of Athens, Athens, Greece
e-mail: stefania.delgaudio@unina2.it
We extracted DNA from some Greek skeletons collected in the Museum of Anthropology of the University of
Athens. Real-time quantitative PCR was used to estimate mitochondrial (mtDNA) and nuclear DNA content.
While mtNA was detected and quantified in all samples, amplification of nuclear targets, as amelogenin
(AMG) and polymorphism M470V of cystic fibrosis transmembrane conductance regulator (CFTR) gene,
yielded positive results in one case only.
In order to enhance amplification sensitivity we applied, for the first time in ancient DNA analysis, a
preamplification strategy based on TaqManPreAmp Master Mix (PreAmp). This procedure utilizes a pool
of primers targeting genes of interest and is followed by real-time PCR specific for each target. Our results
showed a significant improvement of amplification sensitivity allowing sex determination and M470V typing
for most samples. We used as control DNA extracted from some of the skeletons collected in Pompeii
archaeological site, that already gave positive results when analysed with a standard PCR amplification for
mtDNA and AMG gene. In Pompeian samples preamplification technique confirmed results obtained in non
preamplified extracts.
This strategy will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.
Keywords: ancient DNA, preamplification, Real-time PCR