Figure 2
From: Digital detection of endonuclease mediated gene disruption in the HIV provirus
Droplet digital PCR can detect a wide range of deletion mutations.
(A) Primer and probe design. HIV pol-specific forward and reverse primers are used with a reference (1) and target (2) probe that bind to opposite strands of the same PCR amplicon. The target probe (2) is centered on the megaTAL target site, indicated by a red triangle. (B) Target site deletion mutations used for ddPCR assay validation. (C) Two dimensional ddPCR amplitude plot showing that the assay detects the reference sequence in addition to wild type target sequence (X), one base pair deletions (Y), or 2, 3, 4 and 7 base pair deletions (Z) at the megaTAL target site in reference plasmids. Mutant control plasmids were spiked into a background of wild type plasmid at an approximate ratio of 85:1:2 (X:Y:Z). Droplets containing no target are shown in gray. (D) Two dimensional ddPCR amplitude plot showing that the assay detects the reference sequence, wild type (X) and mutant (Y, Z) sequences in 293T cells 72 hours after transfection with pDHIV3 and plasmids expressing the 6.5 megaTAL and Trex2. (E) 293T cells with pDHIV3 and no megaTAL treatment (negative control) have a very low false mutation rate; >99% of reference positive droplets are also target positive (X).
