|Title:||An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases||Authors:||Huang, S.-H.
JYH-MING JIMMY JUANG
ERIC YAO-YU CHUANG
|Issue Date:||2018||Publisher:||Royal Society of Chemistry||Journal Volume:||143||Journal Issue:||6||Start page/Pages:||1367-1377||Source:||Analyst||Abstract:||
In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice. ? 2018 The Royal Society of Chemistry.
|ISSN:||0003-2654||DOI:||10.1039/c7an01648d||SDG/Keyword:||oligonucleotide; SCN5A protein, human; sodium channel Nav1.5; DNA microarray; dna mutational analysis; genetics; heart arrhythmia; human; microfluidic analysis; nucleic acid hybridization; point mutation; Arrhythmias, Cardiac; DNA Mutational Analysis; Humans; Microfluidic Analytical Techniques; NAV1.5 Voltage-Gated Sodium Channel; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Point Mutation
|Appears in Collections:||醫學系|
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