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

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.