Screening for CGG Repeat Expansion in the FMR1 Gene by Melting Curve Analysis of Combined 5′ and 3′ Direct Triplet-Primed PCRs

Teo, Clara R.L.; Law, Hai Yang; Lee, Caroline; Chong, Samuel S.

doi:10.1373/clinchem.2011.174615

Cited by 26 publications

(38 citation statements)

References 40 publications

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“…The kit was developed on the basis of a previous study. 29 It uses a direct TP-PCR, which includes a combination of primers that target a flanking region and from within the CGG repeats. The primer mix was designed to amplify from the 3 0 end of the FMR1 CGG repeat region of non-modified genomic DNA.…”

Section: Tp-pcr Assay With Mcamentioning

confidence: 99%

“…The primer mix was designed to amplify from the 3 0 end of the FMR1 CGG repeat region of non-modified genomic DNA. 29 The required PCR mixture and DNA polymerase were also included in the kit.…”

Section: Tp-pcr Assay With Mcamentioning

confidence: 99%

“…The underlying technology was developed on the basis of a previous study by Teo et al 29 The assay uses a direct TP-PCR Q8 , coupled with melting curve analysis (MCA) for detecting CGG expansions. The melting temperatures of the PCR amplicons are determined from the melt curve profile and depend on amplicon size.…”

mentioning

confidence: 99%

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Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Lim¹,

Loo²,

Mundhofir

et al. 2015

The Journal of Molecular Diagnostics

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Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. On the basis of testing with well-characterized DNA samples, the assay gave a detection limit of 10 ng per reaction and an analytic specificity beyond 150 ng per reaction. Furthermore, the melt temperatures critical for result interpretation were found to be closely linked to the CGG expansion lengths with great consistency (CV < 0.55%). The clinical performance of the assay was established with 528 blinded and previously analyzed clinical samples, yielding results of 100% sensitivity (95% CI, 91.0%-100%) and 99.6% specificity (95% CI, 98.5%-99.9%) in detecting expansions >55 CGG repeats in FMR1. This new approach eliminates post-PCR handling for all non-expanded samples, and exemplifies a truly efficient screening procedure.

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Section: Tp-pcr Assay With Mcamentioning

confidence: 99%

Section: Tp-pcr Assay With Mcamentioning

confidence: 99%

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Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Lim¹,

Loo²,

Mundhofir

et al. 2015

The Journal of Molecular Diagnostics

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“…21 TP-PCR amplicons are almost always analyzed by CE, although TP-PCR products of the FMR1 CGG repeat have also been analyzed on agarose gels 28 and by MCA. 27,29 Although fluorescent fragment analysis by CE is not prohibitively expensive, MCA may represent a more costeffective rapid screening tool to exclude DM1 CTG repeat expansion in patient samples, thus confining confirmatory CE to only screen-positive samples.…”

Section: Discussionmentioning

confidence: 99%

“…This was automatically followed by MCA of TP-PCR products as previously described. 27 Melt curves and melt peaks were generated using on-board software, and melt peak temperatures (T m s) were rounded off to the nearest 0.05 C.…”

Section: Tp-pcr and Mcamentioning

confidence: 99%

Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay

Lian

Rajan‐Babu

Singh

et al. 2015

The Journal of Molecular Diagnostics

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Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in vitro fertilization-preimplantation genetic diagnosis outcome. We optimized triplet-primed PCR and melting curve analysis on 17 DNAs from DM1-affected/unaffected cell lines. A blinded test was performed on 60 genotype-known clinical samples. Plasmid constructs pDMPK(CTG)35 and pDMPK(CTG)48 were used to establish threshold temperatures separating DM1-affected from unaffected samples. Postscreen triplet-primed PCR amplicon sizing was achieved by short-cycle labeled-primer extension followed by capillary electrophoresis. Triplet-primed PCR melting curve analysis melt peak temperatures of unaffected and DM1-affected samples were lower and higher than the control plasmids' melt peak temperatures, respectively. Capillary electrophoresis of post-melting curve analysis amplicons was completely concordant with the screening results. Triplet-primed PCR melting curve analysis is a simple and cost-effective screening tool for rapid identification of DM1. The companion confirmation protocol allows quick determination of CTG repeat size when required. This strategy avoids the need to perform capillary electrophoresis sizing on all test samples, limiting capillary electrophoresis analysis to only a subset of cases that are screen-positive.

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High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis

Butterfield

Imburgia

Mayne

et al. 2021

Molec Gen & Gen Med

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Background Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet‐primed PCR (TP‐PCR) is technically challenging and cost prohibitive for population surveys. Methods Here, we present a high throughput, low‐cost screening tool for CTG repeat expansions using TP‐PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. Results We determined that multimodal melt profiles from the TP‐PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri‐modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. Conclusion We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large‐scale testing programs such as population studies and newborn screening programs.

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Screening for CGG Repeat Expansion in the FMR1 Gene by Melting Curve Analysis of Combined 5′ and 3′ Direct Triplet-Primed PCRs

Cited by 26 publications

References 40 publications

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay

High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis

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