Long interspersed element 1 (L1) is a human retrotransposon with over 500,000 copies per genome. Most L1 elements are nonfunctional, meaning they are immobile because of truncations and mutations. Although normal cells can inhibit L1 retrotransposition, ~100 L1 elements are potentially active in germline and somatic tissues. These retrotransposition-competent L1 elements are ~6 kb in length and encode 2 proteins essential for mobilization. L1 insertion, through a mechanism called target-primed reverse transcription, often results in target-site duplications.
Not surprisingly, L1 mobilization has resulted in de novo cases of human disease, for example, diabetes and hemophilia, and is associated with various cancers, including some breast, colon, lung, prostate, liver, renal, bladder, and ovarian cancers. L1-mediated gene expression or retrotransposition may drive tumorigenesis in some cases or may contribute to aberrant gene expression during tumor development.
High throughput sequencing technology has been instrumental in detecting L1 insertion events. However, validation of these de novo insertions can be problematic, due to tumor heterogeneity and the high copy number of L1 elements. In this study, White et al. describe how they designed primers and PrimeTime® qPCR Probes from IDT to maximize PCR amplification of the L1 of interest. They then used droplet digital PCR (ddPCR) to detect and quantify the targeted L1. In each reaction well of a ddPCR experiment, a 5′-nuclease PCR assay is partitioned into ~20,000 droplets as a water-in-oil emulsion. Ideally, each droplet contains 0 or 1 copy of the target DNA, so that the number of fluorescent droplets indicates the number of targets in the reaction. This paper shows how ddPCR allows detection of rare L1 insertion events (as low as an L1 event in 1 in 10,000 cells) and how ddPCR can be used to distinguish heterozygous loci from homozygous loci.