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Gradient optimization of a high-resolution melt (HRM) assay for targeted cancer therapy screening using SsoFast™ EvaGreen® supermix and the CFX96™ real-time PCR detection system
Sponsored,vendor-submitted protocol    Sponsored by Bio-Rad Laboratories    Published in November 2009 (p.37) DOI: 10.2144/000113287


High-resolution melt (HRM) analysis can discriminate nucleotide sequence differences among samples by comparing the differing DNA melting behavior of different sample compositions. HRM offers a cost-effective, yet accurate alternative to probe-based genotyping assays and DNA sequencing (1).

To develop a rapid screening tool to identify K-ras mutations in clinical cancer samples, primers to detect these mutations via HRM were developed (2). KRAS mutations are found in a group of patients that do not respond to EGFR-targeted therapies for adenocarcinomas (3) and have an associated poorer prognosis than those without the mutations (4).

Materials and methods

Genomic DNA from wild-type MCF7 and KRAS mutant A549 and HCT116 cell lines was amplified using SsoFast EvaGreen supermix (Cat. no. 172-5200; Bio-Rad) and primers as described in Reference 2. A 92-bp amplicon covering codon 12 and 13 of the KRAS gene was amplified using the CFX96 real-time PCR detection system, and results were analyzed using the CFX Manager™ and Precision Melt Analysis™ software. Two amplification/melt protocols were compared.

Published protocol

One cycle of 95°C for 15 min; 40 cycles of 95°C for 15 s, 60.7°C for 15 s, 72°C for 90 s, one cycle of 95°C for 1 s, 72°C for 90 s followed by a melt from 72 to 95°C rising at 0.2°C/s.

Optimization protocol

One cycle of 98°C for 2 min; 50 cycles of 98°C for 5 s, thermal gradient ranging from 55 to 70°C for 10 s, one cycle of 95°C for 30 s, 72°C for 30 s followed by a melt from 72 to 95°C rising at 0.2C/s.


Figure 1. Detection of KRAS mutation using HRM assay. (A) With optimal annealing conditions cell lines of differing KRAS genotypes are easily distinguished with confidences >98%. (B) At suboptimal conditions the two KRAS mutant genotypes (black lines) are not individually identifiable. — mutant A549; — mutant HCT116; — wild-type MCF7; — cluster containing both A549 and HCT116 mutants.

The thermal gradient function enabled the optimal conditions for this KRAS mutation HRM assay to be determined in a single run. Using the well groups function of CFX Manager software, each annealing temperature was analyzed independently. It was determined using the Precision Melt Analysis software that the best resolution of the three genotypes was obtained when using annealing temperatures between 60.9°C and -64.5°C. Annealing temperatures outside of this range resulted in less accurate sample clustering and genotype calling, demonstrating the decreased accuracy of results when using suboptimal reaction conditions.

SsoFast EvaGreen supermix and the CFX96 real-time detection system enabled the run time to be shortened to 82 min compared to 120 min for the published protocol run on the Corbett Rotor-Gene 6000, significantly reducing time to results.


1. Wittwer, C.T. et al. (2003). High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853-860.

2. Krypuy, M. et al. (2006). High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer 6:295.

3. Pao, W. et al. (2005). KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2:e17.

4. Keohavong, P. et al. (1996). Detection of K-ras mutations in lung carcinomas: Relationship to prognosis. Clin Cancer Res 2:411-418.

EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium, Inc. to sell reagents containing EvaGreen dye for use in real-time PCR, for research purposes only.

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