Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C. 