Figure 4

TAIL-PCR and integration site identification. a) TAIL-PCR amplification of DNA fragments covering pABr3 integration sites. Representative results obtained from three amplification runs (PCR1, PCR2, PCR3) carried out with three nested primers annealing with the left border (LB) of the T-DNA (LB1, LB2 and LB3) and a degenerate primer annealing with the neighboring genome sequence (AD1) (see explanatory scheme on the right and “Materials and Methods” for details). Template genomic DNA was extracted from a pABr3-transformed mycelium (T) and from a mock-transformed mycelium (WT); size markers (M) are in the leftmost lane. b) Amplification of the right border (RB). c) Integration site sequences retrieved from TAIL-PCR analysis of transformants #2, #8, #10, #11 and #12; The underlined and highlighted in bold sequences represent the portion of the T-DNA adjacent to the unknown sequence obtained by PCR.