J Integr Plant Biol. ›› 2010, Vol. 52 ›› Issue (12): 1036-1042.DOI: 10.1111/j.1744-7909.2010.01001.x

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A Cost-effective High-resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

Yi-Dan Li1, Zhi-Zhan Chu3, Xiang-Guo Liu1, Hai-Chun Jing2, Yao-Guang Liu3* and Dong-Yun Hao1*   

  1. 1Biotechnology Research Centre, Jilin Academy of Agricultural Sciences, Changchun 130033, China.
    2Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China
    3College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
  • Received:2010-05-17 Accepted:2010-09-24 Published:2010-10-05
  • About author:
    **Corresponding authors Tel: +86 431 8706 3195; Fax: +86 431 8706 3080; E-mail: dyhao@cjaas.com Tel: + 86 20 8528 1908; Fax: +86 20 8528 2180; E-mail: ygliu@scau.edu.cn

Abstract:

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.

Li YD, Chu ZZ, Liu XG, Jing HC, Liu YG, Hao DY (2010) A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants. J. Integr. Plant Biol. 52(12), 1036–1042.

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