|Jarret, Robert - Bob|
Submitted to: Handbook of Plant Mutation Screening
Publication Type: Book / chapter
Publication Acceptance Date: 4/12/2009
Publication Date: 1/20/2010
Citation: Mckinney, J.T., Nay, L.M., Dekoeyer, D., Reed, G.H., Wall, M., Palais, R., Jarret, R.L., Wittner, C. 2010. Mutation Scanning and Genotyping in Plants by High Resolution DNA Melting. Handbook of Plant Mutation Screening. pp 149-163. Interpretive Summary: Various techniques are available to identify genetic mutations. In this chapter, a method that monitors DNA melting-reassociation kinetics in a PCR format, for the detection of changes in DNA sequences, is utilized to screen various types of plant materials. Protocols are described in detail in the text. Examples are provided that demonstrate the efficacy of this technology for distinguishing individual plant genotypes, allele dosage in polyploids, and the detection of various species of pepper (Capsicum).
Technical Abstract: High-resolution melting analysis after PCR allows closed-tube mutation scanning and genotyping without processing, labeled probes, real-time monitoring or allele-specific amplification. PCR is performed in the presence of the saturating dye, LCGreen® Plus, with subsequent high-resolution melting analysis on a high-throughput 96 or 384-well LightScanner®. Heterozygotes are easily distinguished from homozygotes by the shape of the normalized melting curves. On the LightScanner, the sensitivity and specificity of detecting SNP heterozygotes is 100% for PCR product lengths from 50-400 bp, and 96.7% and 98.4%, respectively, for 500-800 bps. In contrast to other scanning techniques, no separations are required. Scanning of highly variable segments of diploid organisms differentiates many genotypes, including SNP heterozygotes, double SNP heterozygotes, and homozygotes. In addition to scanning for unknown variants, genotyping of known variants is easily performed with unlabeled probes using the same system. In tetraploid organisms like the potato, unlabeled probes are a nice solution for genotyping with accurate allele dose determination. High-resolution DNA melting can be applied to diploid or polyploid organisms and requires only 5-10 minutes after PCR. Variant detection sensitivity is high and unlabeled probes allow quantitative polyploidy genotyping with a minimum of cost and effort.