ShineRoar探针技术检测单核苷酸多态性

A novel method for detecting single-nucleotide polymorphisms by using ShineRoar probes

目的建立一种低成本、高效率、操作简便的单核苷酸多态性(SNP)检测技术。方法采用自行设计的"ShineRoar 探针",结合融解曲线技术检测目的基因 SNP。并根据其工作原理分别对肿瘤坏死因子受体Ⅱ(TNFRⅡ)和载脂蛋白 M(apoM)的基因多态性进行检测;同时用 DNA 测序技术鉴定 ShineRoar 探针技术的准确性。结果融解曲线分析结果显示,某一基因的野生型及突变型纯合子分别在2个不同的融解温度出现融解谷。TNFRⅡ基因第6外显子196位突变(ATG→AGG)所产生的 T 和 G 等位基因的融解温度分别为(52.84±0.75)℃和(58.38±0.61)℃;apoM T-778C 突变所产生的 T 和 C 等位基因的融解温度分别为(42.55±0.73)℃和(49.19±0.57)℃。一致性 Kappa 检验显示 ShineRoar 探针技术与 DNA 测序技术的 SNP 检测结果一致(Kappa=1,P=0.000)。结论ShineRoar 探针技术简单、快速、准确,适用于大批量基因分型的研究。

Objective The present study demonstrates a novel, simple and cost-effective method for detecting known SNP genotyping by using ShineRoar probes. Methods The SNP of target genes detected by using the ShineRoar probes and melting curve analysis. Tumor necrosis factor receptor Ⅱ （TNFRⅡ） and apolipoprotein M （apoM） had been employed as target genes to describe the method in details. The PCR products of TNFR n and apoM were collected and sequenced. Results The melting temperatures （ TM ） were significantly different between mutated genotypes and wild-type genotype. A biallelic SNP marker （T/ G） at position 196 in exon 6 of TNFR n gene showed two melting valleys with the appropriate TMs at （52.84±0.75） ℃ and （58.38 ±0.61） ℃respectively. For apoM T-778C, TMs of homozygous T genotype and C genotype were （42.55 ± 0.73 ） ℃ and （49.19 ± 0.57 ） ℃ , respectively. Moreover, this genotyping method was validated by the DNA sequence analyses （ Kappa = 1, P = 0.000 ）. Conclusion It is concluded that this novel method is simple and economical and it is suitable for a large-scale genotyping screening.