Eliminating False Positives

Generating High Quality TILLING Images

The LI-COR 4300 DNA Analysis System is uniquely suited for TILLING because it uses two-color infrared fluorescence detection to generate two true gel images during electrophoresis. Unprocessed image data are critical for TILLING because systems that highly process fluorescence data during detection will likely filter out most, if not all, mutations.

Two-Color Imaging Eliminates False Positives

Mutation identification on the 4300 System is very accurate. False positives are virtually eliminated by two-color imaging. Even on the highly reduced gel images shown to the right, bands of mutations are clearly visible (circled red and green). When heteroduplexes created during PCR are cleaved at base mismatches (see TILLING Workflow), the result is two cleavage fragments labeled with different IRDyes. With two color imaging, a true mutation has two mutant bands below the wild type band in the same lane. One band is on the IRDye® 700 infrared dye image and the other on the IRDye® 800 infrared dye image. Additionally, the sum of the molecular weights of the mutant bands in a lane must equal the molecular weight of the wild type band in order for the mutation to be confirmed. Using this two-color detection method, the 4300 System virtually eliminates false positive mutation identifications.

Data courtesy of the Arabidopsis TILLING project, Seattle, Washington.