A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk.

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Presentation transcript:

A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk Populations Flora Tassone, Ruiqin Pan, Khaled Amiri, Annette K. Taylor, Paul J. Hagerman The Journal of Molecular Diagnostics Volume 10, Issue 1, Pages 43-49 (January 2008) DOI: 10.2353/jmoldx.2008.070073 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of the secondary PCR screening method to resolve apparent homozygosity in females. The approach uses a combination of betaine and a chimeric (CGG-targeted) PCR primer that primes randomly, but with a size bias for amplification of smaller expansions, within the CGG repeat. For a normal homozygous female (30,30 CGG repeats) (gel lane 2: c and f primers of Fu et al32; open arrows), only small PCR products are produced with the chimeric primer (gel lane 4: c, open arrow, and chimeric primer; line arrow). However, for a full-mutation female (28, 510, 615 CGG repeats) (lane 3: c and f primers, apparent homozygote), an extensive smear is produced with the chimeric primer (lane 5: c and chimeric primer), reflecting priming within the extended CGG repeat. For full-mutation alleles, priming by the downstream primer (primer f) does not occur. Representation of the PCR products for each lane is given to the right of the gel image (2% agarose). The chimeric primer comprises a 3′-(CCG)4 block for targeting and a 5′ unique N24 block for subsequent amplification [N24-(CCG)4]. Allele sizes of the normal and full-mutation females were obtained by both PCR (on acrylamide gel) and Southern blot analysis as described in Materials and Methods. The Journal of Molecular Diagnostics 2008 10, 43-49DOI: (10.2353/jmoldx.2008.070073) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: 2% agarose gel of PCR products using the chimeric primer. Lanes 1 to 3: DNA templates from three females with full-mutation alleles; lanes 4 to 7: DNA derived from four females with normal alleles; lane 8: control PCR without template DNA. B: Southern blot analysis of DNAs depicted in A. Lanes 2 to 4: full-mutation alleles corresponding to lanes 1 to 3 of A. Lanes 1, 5 to 7: normal alleles (2.8- and 5.2-kb bands) corresponding to lanes 4 to 7 of A. Different markers are used for each panel, and there is no direct size correspondence between gels. The Journal of Molecular Diagnostics 2008 10, 43-49DOI: (10.2353/jmoldx.2008.070073) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 A: PCR product (normal allele of 28 CGG repeats) was detected from 10, 5, 2, 1, and 0.1% (where 1% or 1 μl contains between ∼1 and 5 ng of DNA) (lanes 1 to 5, respectively) of the eluted DNA from a single dried blood spot (∼4 × 8-mm FTA cards; Whatman, Inc.). Elution of DNA was according to manufacturer's instructions; almost no PCR product was detected from 0.01% of the dried spot (lane 6). Alleles from a premutation female (21 and 86 CGG repeats), amplified from 1 and 0.1% of eluted DNA, are shown in lanes 7 to 8. Five microliters of PCR product were loaded per lane. B: a mixture of control (21 CGG repeats) and premutation (∼200 CGG repeats) alleles were amplified using different proportions of DNAs from the control and carrier males. Both bands were visible in lane 1 (100 ng of DNA from each sample) and lane 2 (100 ng of premutation DNA and 50 ng of control DNA) of the 2% agarose gel. A female control (30 and 54 CGG repeats) is also shown (lane C). PCR products obtained from a small portion of a dried blood spot from a control male, with (lane 4) and without (lane 3) a washing step, added directly to the PCR reaction. M: marker (High-Low; Bionexus, Oakland, CA). Tick marks to the right of each figure indicate 300, 400, and 500 bp in A and 300, 500, and 750 bp in B. The Journal of Molecular Diagnostics 2008 10, 43-49DOI: (10.2353/jmoldx.2008.070073) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Flow diagram for screening of DNA or blood spots from 50,000 females. First round PCR screening will size all normal and/or premutation bands and will pass through to a second PCR screen (using c and CGG-repeat targeted chimeric primers), all samples displaying a single band (∼20,000). Nearly all of these samples (∼19,985) will be homozygous normal alleles, with only ∼15 samples passed onto Southern blot analysis, based on the presence of a large smear on the analytical gel. This example is based on an assumed 15 full-mutation alleles per 50,000 samples in the general population. The analysis for males is much simpler; only those samples with no band on the first round are passed to the second PCR screening assay and/or directly to the Southern blot analysis. The Journal of Molecular Diagnostics 2008 10, 43-49DOI: (10.2353/jmoldx.2008.070073) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions