Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting.

Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T(m)). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T(m), as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.