An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene Alessandro Saluto, Alessandro.

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An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene Alessandro Saluto, Alessandro Brussino, Flora Tassone, Carlo Arduino, Claudia Cagnoli, Patrizia Pappi, Paul Hagerman, Nicola Migone, Alfredo Brusco The Journal of Molecular Diagnostics Volume 7, Issue 5, Pages 605-612 (November 2005) DOI: 10.1016/S1525-1578(10)60594-6 Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Effect of betaine concentration on the PCR amplification of the CGG repeat region of the FMR1 gene. Three male subjects, carrying 20, 83, and ∼200 CGG repeats, were tested with increasing concentrations of betaine, from 1.3 to 2.2 mol/L. M, 100-bp ladder molecular weight marker; three marker bands of 300, 500, and 1000 bp are indicated. The Journal of Molecular Diagnostics 2005 7, 605-612DOI: (10.1016/S1525-1578(10)60594-6) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of PCR products from the amplification of the CGG region of the FMR1 gene. Five microliters of PCR product was loaded per lane: a, samples male; b, female samples. CGG genotype is indicated below each lane. M1, 100-bp ladder molecular weight marker. M2, molecular weight marker Hi-Low DNA Marker (Bionexus, Oakland, CA). The Journal of Molecular Diagnostics 2005 7, 605-612DOI: (10.1016/S1525-1578(10)60594-6) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Automated fluorescence analysis of the PCR products for premutation and full mutation subjects. PCR reactions were performed using 1.7 mol/L betaine. Panels 1 and 2, premutation (male) carriers with alleles of 83 and 90 CGG repeats, respectively. Panels 3 to 5, full mutation males; descending array of peaks visible from 240 to 500 bp. Panel 6, full mutation carrier female with 35/>400 CGG repeats; a main offscale peak corresponds to the normal 35-CGG allele, and a set of low-intensity stutter bands is generated by the full mutation. Panel 7, full mutation mosaic male; the profile is similar to that of a full mutation carrier female in panel 6. The Journal of Molecular Diagnostics 2005 7, 605-612DOI: (10.1016/S1525-1578(10)60594-6) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 PCR fragment length (abscissa) versus number of CGG repeats (ordinate). ▴, data from 18 sequenced male subjects and 10 clones (Table 2). ○, derived from the average size of the stutter bands generated in 17 premutation or full mutation male subjects. The Journal of Molecular Diagnostics 2005 7, 605-612DOI: (10.1016/S1525-1578(10)60594-6) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions