Solution-phase DNA mutation scanning and SNP genotyping by nanoliter melting analysis

Abstract

Solution-phase, DNA melting analysis for heterozygote scanning and single nucleotide polymorphism (SNP) genotyping was performed in 10 nl volumes on a custom microchip. Human genomic DNA was PCR amplified in the presence of the saturating fluorescent dye, LCGreen Plus, and placed within microfluidic channels that were created between two glass slides. The microchip was heated at 0.1 degrees C/s with a Peltier device and viewed with an inverted fluorescence microscope modified for photomulitiplier tube detection. The melting data was normalized and the negative first derivative plotted against temperature. Mutation scanning for heterozygotes was easily performed by comparing the shape of the melting curve to homozygous standards. Genotyping of homozygotes by melting temperature (T(m)) required absolute temperature comparisons. Mutation scanning of ATM exon 17 and CFTR exon 10 identified single base change heterozygotes in 84 and 201 base-pair (bp) products, respectively. All genotypes at HFE C282Y were distinguished by simple melting analysis of a 40-bp fragment. Sequential analysis of the same sample on the gold-standard, commercial high-resolution melting instrument HR-1, followed by melting in a 10 nl reaction chamber, produced similar results. DNA melting analysis requires only minutes after PCR and is a simple method for genotyping and scanning that can be reduced to nanoliter volumes. Microscale systems for performing DNA melting reduce the reagents/DNA template required with a promise for high throughput analysis in a closed chamber without risk of contamination.

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detection

“…Meanwhile, DNA melting analysis has drawn great attention as an effective SNP genotyping method because of its excellent detection accuracy without the needs of expensive reagents and complicated procedures. [12][13][14][15][16] As two strands of DNA, or duplex, "melt" when the surrounding temperature rises, the DNA strands, depending on the sequences, have the unique bonding strength and denature at their own melting temperatures (T m ). This melting analysis can serve as a vital technique in today's genotyping and mutation scanning.…”

“…[22][23][24] Liquid phase analysis, on the other hand, eliminates the need of surface modification inside the devices, but it is restricted for one single analysis. 16,25 To address the limitation and to enhance the reaction kinetics, micrometer-sized beads, which functioned as mobile supports, can be utilized. 26 Due to their mobility, these microbeads permit better flow dynamics and mixing efficiency, while preserve a high surface-to-volume ratio on the beads within the system.…”

Rapid prototyping of PMMA microfluidic chips utilizing a CO2 laser

“…Microfluidic chips have attracted significant attention over the past decade due to their wide range of potential applications in the biomedical and chemical analysis fields, including capillary electrophoresis (Fu et al , 2008aOhno et al 2008;Fu et al 2009), flow cytometry (Fu et al 2004(Fu et al , 2008bTsai et al 2008;Wang 2008, 2009;Hou et al 2009;Lin et al 2009), polymerase chain reaction (Prakash and Kaler 2007;Prakash et al 2008;Lund-Olesen et al 2008;Lien et al 2009;Allen et al 2009;Hsieh et al 2009), DNA amplification (Sundberg et al 2007;Lin et al 2008), and protein analysis (Choi et al 2008;Zhang et al 2008a, b;Lee et al 2009;Tran et al 2010). Moreover, many researchers have demonstrated the feasibility of utilizing micromachining techniques to fabricate a network of microchannels on single quartz, glass, or plastic (polymethyl-methacrylate (PMMA), polydimethylsiloxane (PDMS), PC) substrates so as to create microchips capable of performing multiple procedures, e.g., sample handling, mixing, pretreatment, chemical reaction, separation, and so forth (Lee et al 2006;Lin et al 2007;Tsai et al 2007;Beyor et al 2008;Wu and Li 2008;Fu et al 2008c;Zhu et al 2009;Wen et al 2009;Hairer and Vellekoop 2009;Yeh et al 2010).…”

A multilevel Lab on chip platform for DNA analysis

“…The possibility to implement a complex genetic protocol, such as Single Nucleotide Polymorphism (SNP) detection, on microdevices has been previously investigated demonstrating the compatibility between materials and reagents Podder et al 2008;Pullat et al 2008;Sundberg et al 2007). Nevertheless a true LOC approach was not implemented.…”