Anchor Mediated PCR for targeted NGS
There are many applications that require the sequencing of unknown genomic regions. This is an inherent difficulty in targeted genomic sequencing, since traditionally you must know a sequence to design capture methods for it. However it is possible to target many unknown regions based on a known anchor or marker. For example, traditional cloning of upstream of downstream genes by chromosomal walking, finding junction sequence of viral integration sites or transposons, and identifying binding/interacting partners of known proximal or distal genomic elements in the higher order structure as is done in 3C (chromosome conformation capture) experiments. Linker-mediated PCR has been used successfully for this purpose, in which DNA is first digested/fragmented and then ligated to a double stranded linker, followed by PCR with one primer from the known anchor and the other from the linker. The development of high-throughput methods based on next-generation sequencing platforms will allow these experiments to be done on a whole genome scale and will inform many areas of research.