Two samples of bone removed from the ileum, and one of faecal matter from the rectum were digested in temporally separated extractions (4 ml total volume) by a previously published protocol . One extraction blank was produced for each sample, and PCRs also included negative controls.
The specialist Ancient Biomolecules Centre (ABC) at Oxford was used to extract DNA, and set up PCR reactions. The ABC is physically isolated, subject to stringent anti-contamination procedures, equipped with positive air pressure and UV lighting, and has a DNA laboratory and equipment (glove box, instruments, full body suits, protective masks, etc.) dedicated solely to ancient human specimens. The thermal cycling reactions and subsequent downstream work took place in a separate laboratory located in the Department of Zoology.
Initial singleplex PCRs on the control region were conducted with primers listed in additional file 4 and cloned by previously published protocols . Several hierarchical SNPs from the human mtDNA phylogeny together with haplogroup K specific SNPs were identified [13, 40] using additional singleplex PCR primers designed to be compatible under multiplex PCR conditions .
The three control region SNPs, located in the singleplex PCRs, were combined with key SNPs from major clades of the hg K phylogeny, to form the K1 multiplex. A selection of higher order hierarchical SNPs from the human phylogeny were also included to check the consistency of results across additional loci [13, 40]. This integration of all diagnostic sites into a single assay reduces the range of explanations required when results do not conform to expectations because the possibility of sample mix up is eliminated.
The initial multiplex PCR was performed using 2 μl of DNA extract in a 25 μl reaction volume using primers listed in additional file 5 by a previously published protocol . SBE reactions were performed in a total volume of 5 μl with 0.5 μl purified PCR product, 2.5 μl SNaPshot™ reaction mix (AB) and 0.5 μl SBE primer mix (SBE primers listed in additional file 6). Negative controls were included in both stages of the assay to assess the potential for contamination.
Automated allele calls were made using macros constructed in Genotyper 3.7 (AB). Primer concentrations were adjusted to give balanced amounts of products and peak heights, concentrating on the SBE reaction . All primers were synthesized and reverse-phase HPLC purified by Biomers.net GmbH (Ulm, Germany). A detailed protocol for the optimization of SBE multiplexes is available elsewhere .
Quantitative PCR (qPCR) was conducted on the HB50 aDNA bone extract and a modern DNA sample (belonging to the mitochondrial hg J) in 25 μl reaction volumes, using SYBR Green PCR master-mix (AB), 300 nM human-specific primers, synthesized template standards (see additional file 7) and an ABI Prism 7000 Sequence Detection System (95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 60 sec). All samples were run in triplicate with two 1:4 serial dilutions. The standards were also run in triplicate with 1:9 dilutions ranging from 106-102 copies per μl. The adjusted R2 value of all assays performed was equal to a minimum of 99%, and PCR efficiency, in each case, was calculated with the formula
 to be ~2 (see additional file 2).
Once the effective copy number of the modern DNA was known, a range of dilutions were run against the HB50 extract in a relative quantification, using the same primers and conditions, until the reported copy number of both the ancient and modern extracts were equivalent. The multiplex contamination experiments were then performed using successive serial dilutions of the hg J contaminant mixed with constant quantities of the aDNA extract to seed the multiplex PCR. This procedure provides a detailed measure of the sensitivity of the SBE methodology for identifying and estimating the amount of contamination present in an aDNA extract through the incorporation of additional peaks at segregating sites.
All contamination experiments, thermal cycling reactions and downstream work took place in a separate laboratory located in the Department of Zoology. All relevant personnel in this study had their mtDNA typed. The two researchers who took the samples from the Iceman have mtDNA matching hg H and hg J1b. All aDNA work in Oxford (extractions, singleplex and multiplex PCRs) were carried out by PE, who is hg H.
Figures were prepared using Inkscape Vector Illustrator compiled for use in Ubuntu Linux. The real-time PCR plot was created using the R statistical package http://www.r-project.org. The manuscript was written using the LyX document preparation system http://www.LyX.org.