Spatial DNA melting analysis for genotyping and variant scanning

A continuous-flow, temperature gradient microfluidic device was used to demonstrate spatial DNA melting analysis with the resolution and reproducibility necessary for clinical SNP scanning and genotyping of human genomic DNA. With a steady-state temperature gradient of 20-30 degrees C across a sample, melting curves were constructed from a single fluorescence data acquisition. This technique was used to scan for heterozygotes and to fully genotype single base changes using unlabeled probes. Signal-to-noise ratios of 150-300 were achieved. The thermal effects of sample flow were examined, and temperature control was aided by inclusion of an isothermal channel inlet and thermal relaxation times in the experimental protocol. Human single base variants examined by spatial DNA melting analysis included rs354439, HTR2A 102T > C, and three alleles that affect appropriate warfarin dosage (CYP2C9*2, CYP2C9*3, and VKORC1 1173C > T). Heterozygote scanning was demonstrated with rs354439, while the other PCR targets were genotyped using unlabeled probes with T(m) differences of approximately 5 degrees C between genotypes. To validate the method, 12 blinded DNA samples were genotyped at the three warfarin-related sites by spatial DNA melting analysis with 100% accuracy.