Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy

G Trivedi, Pooja ; Gajera, Jaydeep ; I Ghanchi, Fesal ; M Sindhav, Gaurang

Abstract

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA)signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3–0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics.

Keyword(s)

Electrophoresis; Multiplex PCR; Optimization; Physico-chemical indices

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