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Curr Issues Mol Biol. 2009;11 Suppl 1:i1-9. Epub 2009 Feb 2.

A high-resolution melting approach for analyzing allelic expression dynamics.

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1
Potato Research Centre, Agriculture and Agri-Food Canada, Fredericton, NB, E3B 4Z7, Canada.

Abstract

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 distinguish alleles among potato (Solanum tuberosum) transcript abundances. Transcript properties of genes encoding seven carbohydrate metabolism enzymes/proteins in various tissues and cold storage durations were studied. The HRM assay measured 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 and therefore can serve as functional allele activity (FAA) markers. By combining the allelic specificity of HRM with simple PCR design, this technology can be applied to rapidly determine the most active allele of a gene among the cells analyzed.

PMID:
19193959
[Indexed for MEDLINE]
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