Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer

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• 作者：Masamune Aihara^a ; Shigeru Yamamoto^b ; Hiroko Nishioka^a ; Yutaro Inoue^a ; Kimikazu Hamano^c ; Masaaki Oka^b ; Yoichi Mizukami^a ; ^{mizukami@yamaguchi-u.ac.jp}
• 关键词：GPR30 ; G protein-coupled receptor 30 ; GPER-1 ; G protein estrogen receptor-1 ; ORF ; open reading frame ; HRM ; high-resolution melting ; SNP ; single nucleotide polymorphism ; Expand Hi Fi ; Expand High FidelityPLUS Taq DNA polymerase ; Platinum Taq ; Platinum Taq
• 刊名：Gene
• 出版年：2012
• 期刊代码：73_03781119
• 类别：bio
• 出版时间：15 June, 2012
• 卷：501
• 期：2
• 页码：118-126
• 文件大小：1323 K

摘要

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg²⁺ concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity^PLUS and SYTO9 in the presence of 2.0 mM MgCl₂ produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF.