Subfamily-Selective PCR primers for the Human LINE1 L1PA Lineage

Long interspersed nuclear element-1 (LINE1) retrotransposons are classified into different subfamilies based on evolutionary history. For human biology, the L1PA lineage of LINE1s is particularly significant. This lineage contains both retrotransposition-competent and inactive subfamilies. PCR-based methods are widely used to monitor LINE1s in genomic DNA. However, PCR analysis distinguishing between L1PA lineage subfamilies of different evolutionary age is thus far challenging due to the difficulty to design primers that are sufficiently specific. Here, we developed and applied a workflow to design PCR primers that discriminate between different subfamilies of the L1PA lineage. Amplicon sequencing after PCR amplification of genomic DNA confirmed that the primers differentiate between groups of L1PA subfamilies of different evolutionary age. We validated the primers using RT-qPCR and verified consistency with publicly available RNA-seq data. Moreover, inhibition of DNA methyltransferase activity produced a dose-dependent increase in signals from primers selective for the younger subfamilies, consistent with transcriptional de-repression. These primers enable PCR-based surveys that can stratify expression, copy number or epigenetic modification for L1PA subfamilies of differing evolutionary age. Amplicon sequencing of LINE1 L1PA lineage subfamily-specific PCR primers using genomic DNA from human liver.