A high-resolution melting approach for analyzing allelic expression dynamics.
Yuan, J.Z., Haroon, M., Lightfoot, D.A., Pelletier, Y., Liu, Q., and Li, X.-Q. (2009). "A high-resolution melting approach for analyzing allelic expression dynamics.", Current Issues in Molecular Biology (CIMB), 11(Suppl. 1), pp. i1-9.
Single nucleotide polymorphisms (SNPs) are single base pair mutations that provide new approaches to studies of allele transcript abundances. High-resolution DNA melting curve (HRM) analysis using a LightScanner (Hi-Res Melting™ system with Idaho’s LC Green) provides post-PCR detection of mutations and SNPs in genomic DNA. This study investigated whether the HRM analysis can sensitively profile potato (Solanum tuberosum) transcript abundances and reflect the transcript properties of genes encoding seven carbohydrate metabolism enzymes/proteins in various tissues and cold storage durations. The HRM assay profiled the differential expression of alleles between different organs, between different storage treatments and stages of tubers from the same variety, and between different varieties with the same treatment. The RT-PCR amplicons were directly sequenced to assist the interpretation of HRM data. The cDNA HRM curves correlated well with the nucleotide polymorphisms of the cDNA sequences and the transcript abundance of alleles. By combining the allelic specificity of HRM with simple PCR design, this technology can be applied to rapidly analyze the allelic activity dynamics and determinate the most active allele of a gene in cells analyzed.
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