摘要
目的:建立ABCG2 34G>A和421C>A单核苷酸多态性位点的焦磷酸测序方法。方法:抽取志愿者静脉血入EDTA抗凝管,常规苯酚/氯仿法制备全血gDNA,生物素标记引物对扩增多态位点目的片段,制备生物素标记单链模板,与测序引物退火结合后行焦磷酸测序。分析结果经毛细管电泳测序验证,并进行重复性检验。结果:本文建立了针对ABCG2 34G>A和421C>A多态性位点的焦磷酸测序方法,经毛细管电泳测序验证和重复性验证,结果准确可靠。ABCG2 34G和34A等位基因频率分别为79.5%和20.5%;421C和421A等位基因频率分别为72.7%和27.8%,均符合Hardy-Weinberg平衡。结论:本文建立的焦磷酸测序方法可准确、高通量、快速检测ABCG2 34G>A和421C>A单核苷酸多态性,并且特别适宜大样本量的临床及科研批量检测需要。
AIM： To establish a pyrosequenc- ing based method for detection ABCG2 34G〉A and 421C〉A polymorphisms and to determine the frequency of these polymorphisms in healthy Chinese. METHODS： After preparation of gD- NA from blood of 200 subjects, the target frag- ments were amplified by PCR, polymorphisms were detected on PyroMark ID by pyrosequenc- ing technology. The reliability of pyrosequenc- ing methods were validated by repeat tests and Sanger sequencing. RESULTS： We established a new pyrosequencing method to detect the AB- CG2 34G〉A and 421C〉A polymorphisms poly- morphisms in healthy Chinese. The detection rate and repetition rate were both 100%. The frequencies of ABCG2 34G and 34A alleles were 79.5% and 20.5 % respectively. The allele fre- quencies of ABCG2 421C and 421A were 72.7%and 27.8%, respectively. Genotype frequencies match the Hardy-Weinberg equilibrium. CON- CLUSION： These pyrosequencing assays to de- tect ABCG2 polymorphisms are proved to be a rapid, accurate and high-throughput alternative to conventional methods, and it can be a pre- ferred option in research and clinical application.
出处
《中国临床药理学与治疗学》
CAS
CSCD
2012年第7期779-784,共6页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
国家自然科学基金项目(81072706
81173134)
高等学校博士学科点专项科研基金资助课题(20090162120024)
湖南省科技计划项目(2009JT3020)
中央高校基本科研业务费(2012QNZT085
2012QNZT133)
