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New PCR Assay Rapidly Diagnoses Myotonic Dystrophy Gene Targets

By LabMedica International staff writers
Posted on 01 Mar 2015
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Streck\'s real-time PCR PhilisaW Thermal Cycler for faster time-to-results
The real-time PCR PhilisaW Thermal Cycler for faster time-to-results (Photo courtesy of STRECK LABORATORIES)
In a proof-of-principle study, scientists have developed a conventional-PCR diagnostic test that specifically identifies myotonic dystrophy type 1 (DM1) gene targets from whole blood samples in 15 minutes, obviating the need for prior DNA purification or concentration.

Many PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. However, direct PCR protocols have also traditionally employed thermal cyclers with slow ramp-rates and conservative hold-times. The objective of the study was to reduce sample preparation and assay time for a PCR-based genetic test by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler for direct analysis of whole blood samples.

Scientists from Streck, Inc. (Omaha, NE, USA) improved a genetic screening assay of DM1, a triple-repeat genetic disorder and the most common adult form of muscular dystrophy, through adaptation of a conventional PCR by using Streck’s “PhilisaW Thermal Cycler” and New England Biolabs’ inhibitor-resistant “NEB NextW High-Fidelity 2X PCR Master Mix.” Template sample was 10% whole blood. For detection agarose gel electrophoresis or an Agilent 2100 Bioanalyzer was use. As a reference assay, Gene Link’s “Myotonic Dystrophy Genemer Kit” was used per the kit-specific PCR protocol.

PCR amplification of the DM1 short tandem repeats was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, 23 (57.5%) were identified as DM1 negative. These results were 100% concordant with results using the reference kit.

The outcome is a simple exclusionary screening assay for DM1, independent of up-front sample prep, with significant improvement in time-to-results. This approach could also be applied to adapt other conventional PCR tests where genomic DNA is targeted for analysis.

The paper, by Connelly C et al., was published in the December 2014 issue of the journal BMC Medical Genetics.

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