Figure 2.

C9orf72 (G₄C₂) repeat expansion detection using repeat-primed PCR. Genomic DNA was amplified using a three-primer system and touchdown PCR cycling program as detailed in the methods and previously described (22,23). In brief, the forward primer contains a 5′ fluorescently labeled tag that hybridizes upstream of the (G₄C₂) repeat expansion, the reverse primer consists of a non-complementary 5′ tail sequence and three (G₄C₂) repeat units, and the anchor primer corresponds to the anchor sequence of the reverse primer. As shown in the representative electropherogram, the initial peak corresponds to the shortest fragment containing a minimum of three (G₄C₂) repeats, with each successive peak corresponding to fragments expanding by one repeat unit. A characteristic saw-tooth pattern is generated as signal intensity decreases as fragment size increases. Electropherograms displaying ≥30 repeats is commonly used as the pathological cut-off value (23).