Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer
Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer
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DOI：
10.1016/j.gene.2012.04.029
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发表时间：
2012-06-15
期刊：
影响因子：
3.5
通讯作者：
Mizukami, Yoichi
中科院分区：
文献类型：
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作者：
Aihara, Masamune;Yamamoto, Shigeru;Mizukami, Yoichi
G蛋白偶联受体30/G蛋白雌激素受体-1 （GPR30/GPER-1）是一种新型的雌激素膜受体，其mRNA在雌激素依赖细胞如乳腺癌细胞系中高水平表达。然而，与疾病相关的GRP30突变仍未报道。为了快速检测GPR30开放阅读框（ORF）中的未知突变，研究了高分辨率熔融（HRM）分析的实验条件，包括PCR引物、Taq聚合酶、饱和DNA结合染料、Mg2+浓度和归一化温度。在最佳实验条件下，检测了GPR30 ORF中已知的9个snp和13个人工点突变，以及从乳腺癌受试者中提取的DNA中的单核苷酸变异。在2.0 mM MgCl2存在下，使用PLUS和SYT09对GPR30 ORF各区域突变的熔解曲线分离效果最好。在这些实验条件下，突变在杂合子和纯合子中都被清楚地检测到。利用乳腺癌患者的基因组DNA对GPR30进行HRM分析，发现GPR30中有一个新的单核苷酸变异，111C>T和4个已知的snp。本研究确定的HRM分析实验条件对于检测GPR30 ORF内未知突变的高通量分析是有用的。(C) 2012 Elsevier B.V.版权所有
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 SYT09 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. (C) 2012 Elsevier B.V. All rights reserved.