Standardization of PCR amplification for fragile X trinucleotide repeat measurements*

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Abstract
To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab‐PAGE and capillary (CE) separation systems (b) interlane variability (slab‐PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using GeneScan™ analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab‐gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab‐PAGE and CE size measurements were in agreement except for the pre‐mutation alleles, which yielded significantly smaller sizes by CE.