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Rapid, real-time chemiluminescent detection of DNA mutation based on digital microfluidics and pyrosequencing

Author:
Zou, Fenxiang, Ruan, Qingyu, Lin, Xiaoye, Zhang, Mingxia, Song, Yanling, Zhou, Leiji, Zhu, Zhi, Lin, Shuichao, Wang, Wei, Yang, Chaoyong James
Source:
Biosensors & bioelectronics 2019 v.126 pp. 551-557
ISSN:
0956-5663
Subject:
DNA, automation, bioluminescence, biosensors, genes, genetic testing, microfluidic technology, mutants, mutation, sequence analysis, sex determination analysis
Abstract:
To explore genome mutation meaningfully, it is in urgent need to develop an automated and inexpensive platform for DNA mutation analysis. Digital microfluidics is a powerful platform for a broad range of applications due to the advantages of high automatization and low reagent consumption. Pyrosequencing enables DNA sequencing based on non-electrophoresis bioluminescence, which is suitable for rapid and sensitive analysis of short sequences. Herein, we describe a palmtop sequencing platform for automatic, real-time and portable analysis of DNA mutations, which is based on the pyrosequencing principle and implemented by digital microfluidics. The portable system can sequence a DNA template with up to 53 bp with 100% accuracy within 2 h. Mutation in the KRAS gene can be detected within 30 min with a LOD as low as 5% mutant level. Portable and accurate gender identification was further demonstrated by sequencing a short amelogenin fragment. With the advantages of portability, ease of use, high accuracy, and low cost, the palmtop sequencing platform shows great potential for portable genetic testing in a variety of circumstances.