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Nature Protocols:SNP新型测定方法

来源:生物谷 2009-06-19 17:23

单核苷酸多态性(简称SNP)是导致不同个体间生物学性状(如疾病的易感性、临床转归和药物治疗的敏感性等)差异的遗传学基础,是造成遗传性疾病的主要原因之一,有望成为预测疾病发生、病程进展、治疗效果以及预后的重要遗传标记。但是,SNP的快速、准确分型仍是当前迫切需要解决的重大技术问题。

北京蛋白质组研究中心周钢桥研究员课题组与中国科学院化学研究所王树研究员课题组合作,发展了一种联合运用水溶性共轭聚合物(简称CCP)和单碱基引物延伸反应技术的新的SNP分型方法。该方法既利用单碱基引物延伸反应具有特异性的优势,又利用CCP 具有的高效荧光共振能量转移(简称FRET)所产生的高敏感性的独特优点,大幅提高了SNP基因型分辨的特异性和敏感性。与现有的基于凝胶电泳、普通荧光和质谱等技术的SNP分型方法相比,该方法还具有无需引物标记、操作更简便和无需昂贵检测仪器等优点。优化后的SNP分型实验全程仅需5.5-7.5小时左右。上述研究成果对于提高SNP分型技术的效率、扩展水溶性共轭聚合物的应用范围等均有重要意义,将有力促进我国人群疾病易感性等的研究和防治。相关工作已在线发表于《自然-实验手册》上。(生物谷Bioon.com)

生物谷推荐原始出处:

Nature Protocols 4, - 984 - 991 (2009) doi:10.1038/nprot.2009.70

Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution

Xinrui Duan1,4, Wei Yue2,4, Libing Liu1, Zhengping Li3, Yuliang Li1, Fuchu He2, Daoben Zhu1, Gangqiao Zhou2 & Shu Wang1

This protocol describes a simple, convenient and sensitive single-nucleotide polymorphism (SNP) genotyping method using an optically amplifying cationic conjugated polymer and single base primer extension reaction. The fluorescence resonance energy transfer (FRET) efficiency between the conjugated polymer (PFP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide}) and a fluorescein-labeled dNTP (dNTP-Fl) is correlated to the incorporation of the dNTP-Fl into an allele-specific primer; incorporation occurs by a single base extension reaction when the target DNA and the primer are complementary at the SNP site. By triggering the FRET from PFP to fluorescein and measuring the change in fluorescence intensity of samples, the SNP genotypes can be discriminated. In comparison with other SNP genotyping methods, this protocol simplifies procedures and improves sensitivity by eliminating the need for primer labeling, cumbersome workups, chemical/enzymatic coupling reactions and sophisticated instruments. The assay takes about 2 h for PCR amplification followed by 5.5–7.5 h to obtain the genotypes.

1 Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China.
2 State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.
3 College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei Province, China.
4 These authors contributed equally to this work.

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