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

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Title Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy
 
Creator Trivedi, Pooja G
Gajera, Jaydeep B
Ghanchi, Fesal I
Sindhav, Gaurang M
 
Subject Electrophoresis
Multiplex PCR
Optimization
Physico-chemical indices
 
Description 31-42
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.
 
Date 2023-01-10T10:56:30Z
2023-01-10T10:56:30Z
2023-01
 
Type Article
 
Identifier 0975-0959 (Online); 0301-1208 (Print)
http://nopr.niscpr.res.in/handle/123456789/61189
https://doi.org/10.56042/ijbb.v60i1.61207
 
Language en
 
Publisher NIScPR-CSIR, India
 
Source IJBB Vol.60(01) [January 2023]