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A Cost‐effective High‐resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants
Author(s) -
Li YiDan,
Chu ZhiZhan,
Liu XiangGuo,
Jing HaiChun,
Liu YaoGuang,
Hao DongYun
Publication year - 2010
Publication title -
journal of integrative plant biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.734
H-Index - 83
eISSN - 1744-7909
pISSN - 1672-9072
DOI - 10.1111/j.1744-7909.2010.01001.x
Subject(s) - high resolution melt , genotyping , single nucleotide polymorphism , melting curve analysis , dna , snp genotyping , biology , genetics , polymerase chain reaction , indel , molecular inversion probe , accession number (library science) , primer (cosmetics) , microbiology and biotechnology , genotype , gene , genbank , chemistry , organic chemistry
High‐resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double‐stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real‐time polymerase chain reaction (PCR) and post‐PCR DNA melt curve analysis. Here we report on the development of an EvaGreen‐based HRM analysis and its performance, in comparison with the popular LCGreen‐based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen‐ or LCGreen‐based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1‐3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)‐mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.