Supplemental Figure S2.

Amplification of DNA from challenging templates. A: Amplification of full mutation (FM) alleles from saliva. Genomic DNA was prepared from saliva DNA obtained from male FM carriers FX-1, FX-2, and FX-4, and the repeat size analyzed with and without HpaII digestion, as described in Materials and Methods. The PCR products produced from purified DNA from a sample with 97 repeats (SC120) and 940 repeats (NA09237) are shown for comparison. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-kb molecular weight ladders, respectively. The bracket indicates a smear of products in the FM range that was reproducibly obtained for this sample. This smear is less apparent after HpaII digestion, suggesting that although the premutation (PM) alleles are methylated, the FM alleles are not. Analysis of this DNA by qMS-PCR confirmed that >50% of the alleles in this sample are unmethylated (Table 1). B: Amplification of large methylated FM alleles using the pulse extension modification. Our standard RPT-PCRs were performed using a pulse extension protocol for the amplification, as described in Materials and Methods. The resultant products were resolved by agarose gel electrophoresis, and the DNA visualized using SYBR Gold staining. MW1 is a 1-kb molecular weight ladder. The arrowheads indicate alleles that are only seen using the pulse PCR protocol. Lane 1, Water; lane 2, NA09237; lane 3, C10700; and lane 4, C10259. C: Capillary electrophoretogram of the RPT-PCR products for patient FX-4 demonstrating the presence of FM alleles. M, male.