1. Fragile X Syndrome The Journal of Molecular Diagnostics
Justine I. Lyons, Gregory R. Kerr, Patricia W. Mueller 
The Journal of Molecular Diagnostics 
Volume 17, Issue 5, Pages (September 2015)
DOI: /j.jmoldx
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2. Figure 1
Expansion of the CGG repeats in the 5′ untranslated region of the FMR1 gene on the X chromosome can result in decreased mRNA and FMRP production, causing fragile X. FMRP, FMR1 protein.
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3. Figure 2
Tassone fragile X screening method29: A: The first step uses sequence-based forward and reverse PCR primers to amplify the CGG repeat region fragments which are analyzed on an ABI 3730 capillary electrophoresis DNA analyzer. B: If there are less than two peaks for females or no peak for males, the forward primer is run with a CGG-targeted reverse primer with capillary electrophoresis analysis of the resulting fragments.
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4. Figure 3
The Orpana fragile X screening method29 uses multiple heat pulses in the PCR extension step to destabilize secondary structures and to enhance the extension over the GC-rich sequence of the CGG repeat region. The method uses agarose gel electrophoresis to detect the resulting amplicons.
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5. Figure 4
The Teo fragile X screening method31 uses a melting curve analysis of triplet-primed PCR products in the 5′ and 3′ directions. The repeat-annealing primers tail-CCGR and tail-CGGF anneal fully within the repeat sequence and are tailed at their 5′ ends with noncomplementary sequences to enhance the production of amplicons.
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6. Figure 5
Tassone method.29 A: A normal sample from a heterozygous female and a normal sample from a male analyzed with sequence-based forward and reverse primers. B: Samples from a female and male with a gray zone allele analyzed with sequence-based forward and reverse primers. C: A sample from a female with a normal 29 CGG-repeat allele and an expanded allele, and a sample from a male with an expanded CGG-repeat allele analyzed with sequence-based forward and reverse primers. D: Samples from a homozygous female with two normal 30 CGG-repeat alleles, a sample from a female with a normal and an expanded CGG allele, and a sample from a male with an expanded CGG allele run with a sequence-based primer and a chimeric CGG-targeted primer.
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7. Figure 6
Orpana method.30 Agarose gels of amplicons generated by heat-pulse PCR from males (A) and females (B). The allele sizes are indicated above the tracks.
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8. Figure 7
Teo method.31 Melting curves from a normal sample from a female with 18 and 29 CGG repeats (A) and a full mutation sample from a female with 21 and 650 CGG repeats (B). The vertical reference lines represent cutoffs of 83.3°C for the 5′ assay and 91°C for the 3′ assay.
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9. Figure 8
Teo method.31 The sensitivity and specificity of the 3′ and 5′ assay at different temperature cutoffs.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
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