Directly accessing de novo L1 retrotransposition in the human germline

Human chromosomes are riddled with sequence elements that have in our evolutionary past reproduced and "jumped" to new locations. Long Interspersed Nuclear Element 1 (L1) is the only human autonomous retrotransposon thought to be currently active. L1 retrotransposition has significantly shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 retrotransposition dynamics are little understood in the germline since de novo insertions occur infrequently (one event genome-wide per 16--500 haploid genomes). Also, a lack of germline cell cultures requires techniques that can directly access de novo retrotransposition in sperm DNA.;Hybridisation enrichment was used to capture rare L1 containing DNA from an aliquot of multiplex amplified DNA. Enrichment was achieved by the binding of biotinylated oligonucleotides (bio-oligos) to L1 sequences and their physical retrieval using streptavidin-coated paramagnetic beads. Using bio-oligos on total sperm DNA would be futile due to L1 comprising ∼17% by mass of the genome. The search area was therefore limited to genomic sections (∼5 kb) devoid of matches to the bio-oligo sequences. PCR had to be extremely efficient as a de novo insertion would appear once in a pool of sperm DNA. Multiplex PCR could amplify full length L1 insertions into ∼5 kb target sites to levels of recoverable by hybridisation enrichment from single molecules, despite the presence of overwhelming masses of empty target sites.;After screening ∼600 microg of sperm DNA, I propose a rate (within previous estimates) of <3 insertions in 290 haploid genomes. This low rate of insertion may not be inconsistent with ongoing L1 proliferation. Rather the data supports the idea that L1s may retrotranspose rarely during embryogenesis before germline differentiation. Such "jackpot" insertions colonise numerous gametes, and thus have a greater chance of being inherited, even if this only occurs in very few individuals.