L1 hybridization enrichment: a method for directly accessing de novo L1 insertions in the human germline

Abstract

Long interspersed nuclear element 1 (L1) retrotransposons are the only autonomously mobile human transposable elements. L1 retrotransposition has shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 germline retrotransposition dynamics are poorly understood because de novo insertions occur very rarely: the frequency of disease-causing retrotransposon insertions suggests that one insertion event occurs in roughly 18–180 gametes. The method described here recovers full-length L1 insertions by using hybridization enrichment to capture L1 sequences from multiplex PCR-amplified DNA. Enrichment is achieved by hybridizing L1-specific biotinylated oligonucleotides to complementary molecules, followed by capture on streptavidin-coated paramagnetic beads. We show that multiplex, long-range PCR can amplify single molecules containing full-length L1 insertions for recovery by hybridization enrichment. We screened 600 µg of sperm DNA from one donor, but no bone fide de novo L1 insertions were found, suggesting a L1 retrotransposition frequency of <1 insertion in 400 haploid genomes. This lies below the lower bound of previous estimates, and indicates that L1 insertion, at least into the loci studied, is very rare in the male germline. It is a paradox that L1 replication is ongoing in the face of such apparently low activity. Hum Mutat 32:1–11, 2011. © 2011 Wiley-Liss, Inc.