Preamplification Procedure for the Analysis of Ancient DNA Samples

Abstract

In ancient DNA studies the low amount of endogenous DNA represents a limiting factor that often hampers the result achievement. In this study we extracted the DNA from nine human skeletal remains of different ages found in the Byzantine cemetery of Abdera Halkidiki and in the medieval cemetery of St. Spiridion in Rhodes (Greece). Real-time quantitative polymerase chain reaction (qPCR) was used to detect in the extracts the presence of PCR inhibitors and to estimate the DNA content. As mitochondrial DNA was detected in all samples, amplification of nuclear targets, as amelogenin and the polymorphism M470V of the transmembrane conductance regulator gene, yielded positive results in one case only. In an effort to improve amplification success, we applied, for the first time in ancient DNA, a preamplification strategy based on TaqMan PreAmp Master Mix. A comparison between results obtained from nonpreamplified and preamplified samples is reported. Our data, even if preliminary, show that the TaqMan PreAmp procedure may improve the sensitivity of qPCR analysis.

Comprehensive Analysis of Circulating microRNA Specific to the Liver, Heart, and Skeletal Muscle of Cynomolgus Monkeys

“…Preamplification is useful to enhance the detection of poorly expressed miRNAs, without introducing a detection bias. 31,32 In the present study, the Cq values of the standard curves constructed using preamplified synthesized controls showed almost same values across the assays. This high reproducibility allowed us to perform absolute quantitation using preamplified samples, which resulted in the detection of additional miRNAs.…”

Genomic Analyses of Acute Flaccid Myelitis Cases among a Cluster in Arizona Provide Further Evidence of Enterovirus D68 Role

“…First-strand cDNA synthesis of the total RNA was performed with a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Preamplification, which has been shown to greatly increase sensitivity in complex samples (66, 67), was performed using the TaqMan PreAmp master mix (Thermo Fisher Scientific) with the EV-D68 primers (Table 3) at a final concentration of 5 to 10 nM. Real-time PCR was run on the 7900HT (Thermo Fisher Scientific) in 10-μl reaction mixtures containing 5 μl PerfeCTa FastMix II, 400 nM each forward primer, 600 nM each reverse primer, 200 nM each probe, and 4 μl preamplified template, with denaturation at 95°C for 3 min and 40 cycles of 95°C for 15 s and 60°C for 1 min.…”

Analysis of ancient mtDNA from the medieval archeological site of Amiternum (L'Aquila), central Italy

“…The dimensions of the amplicons correspond to those expected. The same primer pair was used to amplify the DNA of the bone remains; as expected, as the ancient DNA is fragmented (Del Gaudio et al., 2013), there was no amplification (data not shown).…”

“…PCR amplification was carried out using Hybaid PCR Express Thermo Cycler using the kit KAPA2G Fast HotStart ReadyMix 2X (Kbiosystems). Table 2 shows the primers used, some of them have been found in the literature (Caramelli, 2009; Plantinga et al., 2012; Kim et al., 2008; Del Gaudio et al., 2013), others constructed on gene sequences with Primer Express 3.0 software software (Applied Biosystems, USA).…”