Fig. 3

Approaches to PCR validation of insertions. a Agarose gel electrophoresis of a somatic PCR validation. Three lanes are shown: [L] 2-log ladder (NEB), [N] normal DNA, [T] tumor DNA. An upper band marked by a black arrow is present in the tumor but absent in the normal sample which confirms a somatic L1 insertion occurred in the tumor. b Agarose gel of two L1 3’ PCR validations. Five lanes are shown: [L] 2-log ladder (NEB), [F1] forward primer with L1 primer for insertion on 2p16.3, [R1] reverse primer with L1 primer for insertion on 2p16.3, [F2] forward primer with L1 primer for insertion on 9q21.31, [R2] reverse primer with L1 primer for insertion on 9q21.31. For both insertions, only the reverse primer produces a band when paired with the L1 primer, which suggests that both are plus strand insertions. All specific primers were designed approximately 200 bp away from the insertion site. Because the L1 primer is located 150 bp away from the 3′ end of the element, the expected product size for both reactions is approximately 350 bp marked with a gray arrow. The PCR reaction for the 9q21.31 insertion produces a band larger than expected marked with a black arrow. This suggests that a 3′ transduction may have taken place and is confirmed by sending the PCR product for Sanger sequencing. c The illustration shows the relative positions of primers and products for the two L1 insertions from part b. The 9q21.31 insertion in the lower diagram has a 3′ transduction shown as a gold line