Figure 3.

Results of the contamination assay showing the G/A system from three different multiplex SBE reactions (MP1, MP2, MP3), initiated with the Iceman's DNA mixed with three different amounts of an hg J contaminant. Because the contaminant has ancestral alleles at nps 16224, 16311 and 16362, this causes additional peaks to appear in the A system (highlighted). The small peak to the left of 16362A in each case is background noise (i.e. not due to primer length variation which would produce multiple peaks of varying size, see the G system in Figure 2. This does not interfere with the base calling because it is outside the range of either the 00497 or 16362 SBE primers. The sensitivity of this method is demonstrated by its ability to detect the second population of templates (hg J) at the level of ~4%; even at levels of ~20% contamination, the results for the Iceman's mtDNA are unambiguous and unaffected by the contaminant. The ability to test multiple sites in a single reaction, in the presence of contaminants, without the need to clone individual PCR products, represents a significant advantage of the SBE methodology.