DNA Mutation Detection in a Polymer Microfluidic Network Using Temperature Gradient Gel Electrophoresis

Abstract

A miniaturized system for DNA mutation analysis, utilizing temperature gradient gel electrophoresis (TGGE) in a polycarbonate (PC) microfluidic device, is reported. TGGE reveals the presence of sequence heterogeneity in a given heteroduplex sample by introducing a thermal denaturing gradient that results in differences between the average electrophoretic mobilities of DNA sequence variants. Bulk heater assemblies are designed and employed to externally generate temperature gradients in spatial and temporal formats along the separation channels. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, are achieved using both single- and 10-channel parallel measurements in a microfluidic platform. Additionally, a comprehensive polymer microfluidic device containing an integrated microheater and sensor array is developed and demonstrated for performing spatial TGGE for DNA mutation analysis. The device consists of two PC modular substrates mechanically bonded together. One substrate is embossed with microchannels, and the other contains a tapered microheater, lithographically patterned along with an array of temperature sensors. Compared with the external heating approaches, the integrated platform provides significant reduction in power requirement and thermal response time while establishing more accurate and highly effective control of the temperature gradient for achieving improved separation resolution.

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

“…Meanwhile, unconjugated EtBr itself will cause a high-fluorescence background and interferes with the detection sensitivity. 23,44,45 Although our preliminary clinical investigation is limited because of restricted access to more CRC samples, the results of our study showed that the detection accuracy rate reached 100% after verification by clone sequencing. The high accuracy might be due to the relatively high sensitivity of this method, because the mutations harbored in stools can be as little as 1% in general, 11 which is much higher than the threshold concentration (0.2%) of this chip-based TGCE method.…”

“…However, the large external heater assemblies in their spatial format increased difficulty with respect to accuracy control and miniaturization. 23 In our previous work, a chip-based CE platform integrated with a slantwise radiative heating system that was able to establish spatial temperature gradient using simple peripheral equipment and manipulation was developed for very simple, reliable, and cost-effective DNA mutation detection by chipbased TGCE. Various SNP samples and K-ras gene mutations in six colon cancer cell lines were successfully detected rapidly within 6 min.…”

Microwave Dielectric Heating of Fluids in Microfluidic Devices

“…Microwave heating is also very attractive than other alternatives for obtaining spatial temporal temperature gradients for a variety of on-chip applications, including investigation of thermophoresis (Duhr et al, 2006), control and measurement of enzymatic activity (Arata et al, 2005;Mao et al, 2002;Tanaka et al, 2000), investigation of the thermodynamics (Baaske et al, 2007;Mao et al, 2002), chemical separation (Buch et al, 2004;Huang et al, 2002;Ross et al, 2002;Zhang et al, 2007), and of the kinetics characterizing molecular associations (Braun et al, 2003;Dodge et al, 2004). Most techniques for generating on-chip temperature gradients integrate Joule heating elements to conduct heat into microchannels/microchambers (Arata et al, 2005;Buch et al, 2004, Selva et al, 2009).…”

“…Most techniques for generating on-chip temperature gradients integrate Joule heating elements to conduct heat into microchannels/microchambers (Arata et al, 2005;Buch et al, 2004, Selva et al, 2009). However, temporal control is limited by the heat capacity of the microfluidic device and thermal coupling of the device to the heating elements.…”

Low Viscous Separation Media for Genomics and Proteomics Analysis on Microchip Electrophoresis System

“…[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Several types of chips has been introduced for the postgenomic researches such as DNA/RNA chips [28][29][30][31] and the serial analysis of gene expression. [32][33][34][35] The viscosity of the separation media is one important factor for chip separation since the bounded microfluidic devices can not withstand high loading pressure.…”