Figure 3: Schematic representation of experimental steps.
From: Neolithic mitochondrial haplogroup H genomes and the genetic origins of Europeans
(a) Probe DNA was prepared by amplifying a complete mitochondrial genome in two overlapping fragments by long-range PCR, followed by DNA fragmentation and biotinylation to form mtDNA ‘baits’ for targeted hybridisation. (b) Ancient DNA was enzymatically blunt-ended and phosphorylated, ligated to custom library adaptors, followed by polymerase ‘fill-in’ to create ‘immortalised’ double-stranded DNA libraries. (c) Hybridisation-based DNA-capture using biotinylated probe bound to Streptavidin magnetic beads; following stringency washes, captured library constructs enriched in mtDNA sequences are eluted from the beads/probe via a novel polymerase strand-displacement reaction followed by PCR library reamplification. These steps can be carried out iteratively to maximise mtDNA content in enriched libraries (see Supplementary Methods for full details).
