State of Utah Center of Excellence for Biomedical Microfluidics
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| Students involved: Scott Sundberg and Jenny Tayler This work is being done in collaboration with Carl Wittwer in the Department of Pathology. His web page is available at: www.dna.utah.edu Solution-phase, homogeneous DNA melting analysis for heterozygote scanning was performed in 10 nL and 1 nL reaction volumes on a custom microchip and melt curves were in agreement with the gold-standard 10 mL HR-1™ melting instrument. The microchips were characterized for no surface coating and a bovine serum albumin (BSA) coating applied to the microchannels. It was found that BSA improved the reproducibility and S/N for melting analysis. Introduction. In 1997 DNA melting analysis was introduced1, using a double-stranded DNA fluorescent dye to detect single nucleotide polymorphisms (SNPs) and to perform mutation scanning following the polymerase chain reaction (PCR) for DNA amplification. This analysis method is advantageous over other DNA analysis systems because it is less complicated, fast, and prevents contamination of the sample and environment due to its closed-tube technique. Solution-phase or “homogeneous” melting analysis on a microchip is advantageous over microscale systems using hybridized oligonucleotides2 due to the minimization of chip fabrication complexity and reduction in analysis time. Materials and Methods. Xurography3 was used to create the microchips, using a knife plotter to cut out channel structures from double coated tape (9019, 3M, St. Paul, MN) and sandwiching patterned tape between glass slides, Figure 1(A&B). A Peltier heater and J-type thermocouple, with thermal grease at interfaces, were used for temperature control, Figure 1(C). Detection used a modified inverted microscope with optics designed for the double-stranded dye LCGreen® Plus (Idaho Technology, SLC, UT), a photomultiplier tube (PMT) and module, and all hardware was operated using LabView 7.1 (NI, Austin, TX). PCR was performed on the DNA sample and then transferred to an HR-1 instrument for a high-resolution reference melting curve. The sample was then injected into the microchip for melting within 10 nL and 1 nL volumes. Results. Table 1 compares S/N of melts with no microchannel coating and BSA (2.5 mg/mL). BSA coated channels provide a higher S/N and a more reproducible system. Figure 2 shows derivative melting plots of normal and heterozygous DNA for a SNP within an 84-bp fragment of ATM exon 17. Both 1 nL and 10 nL melts are in good agreement with the 10 mL HR-1 melt curves. Temperature shifts between plots is due to a Peltier heater temperature gradient and varying thermocouple placement between microchips. References [1] K. Ririe; R. Rasmussen; C. Wittwer Anal Biochem. 1997, 245, 154-160. [2] A. Dodge; G. Turchatti; I. Lawrence; N. Rooij; E. Verpoorte Anal. Chem. 2004, 76, 1778-87. [3] D. Bartholomeusz; R. Boutte; J. Andrade J. MEMS 2005, 14, 1364-74.
  
 
Table 1. S/N calculations
of microchannels with no coating
Figure 2. Negative derivative plots for the microchip (A:1 nL, B:10 nL) and the HR-1 (C:10 mL) using the ATM exon 17 target. Homozygous wild-type S/N values are 51, 69, and 2450 respectively. Heterozygous mutant S/N values are 59, 63, and 2800 respectively. Homozygous wild-type template (thick line); heterozygous mutant template (thin line). |
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