Figure 3. Expansion levels are affected by the purity of the repeat sequence and the repeat orientation.

(A) PCR analysis of GAA·TTC expansion within an uninterrupted (GAA·TTC)₁₇₆ insert at W0 and W3 compared to a (GAA·TTC)₁₇₆ insert with two interrupting mutations identified by sequencing analysis. An A→T mutation was detected approximately 118 triplets into the repeat region from the 5′ end and a T→G mutation was detected 31 triplets into the repeat region from the 3′ end. PCR amplification adds 438 bp to the GAA·TTC insert. M: 1 Kb plus size standard. (B) PCR analysis of GAA·TTC expansion within an uninterrupted (GAA·TTC)₁₇₆ insert at W0, W2, and W3 compared to a (GAA·TTC)₈₈MfeI(GAA·TTC)₉₀ insert sequence containing an interrupting hexamer sequence located between two repeat tracts. The CAATTG hexamer is the recognition sequence for the Mfe I restriction endonuclease. PCR amplification adds 438 bp to the GAA·TTC insert. M: 1 Kb plus size standard. (C) Mfe I digestion of the PCR amplification products from the (GAA·TTC)₈₈MfeI(GAA·TTC)₉₀ time-course at W0, W2, and W3. PCR amplification adds 338 bp to the 5′ end of the GAA·TTC insert and 100 bp to the 3′ end. Mfe I digestion yields two distinct fragments representing the promoter proximal (GAA·TTC)₈₈ repeat tract (5′ GAA) and the distal (GAA·TTC)₉₀ repeat tract (3′ GAA). The residual full-length product (Uncut) is due to incomplete digestion. M: 1 Kb plus size standard. (D) PCR sizing analysis of a (GAA·TTC)₁₇₆ insert at W0 and W3 compared to a reverse oriented (CTT·AAG)₁₇₆ insert sequence at the same time-points. PCR amplification adds 438 bp to the repeat insert. M: 1 Kb plus size standard. A representative gel from an n = 2 is shown.