摘要
目的应用DHPLC技术对TGFBR3基因12外显子多态进行准确、快速、高通量的检测。方法选择2009年2月至2012年12月在南方医科大学附属深圳市妇幼保健院妇产科确诊的卵巢早衰患者110例为卵巢早衰组,以月经规则、性激素检查在正常范围内的年龄匹配的妇女110例为对照组。针对TGFBR3基因12外显子的编码区及剪接位点设计变性高效液相色谱分析的引物,构建TGFBR3基因12外显子的变性高效液相色谱分析方法。结果建立的针对TGFBR3基因12外显子的变性高效液相色谱分析方法能够快速、准确地区分已知变异序列和野生型序列,并且灵敏性和稳定性都好。收集110例特发性卵巢早衰和110例正常人标本,对这些标本进行TGFBR3基因12外显子的变性高效液相色谱分析检测,检测到2个的单核苷酸多态性((SNP)。2022 T/C位点多态性:卵巢早衰组CC、TC、TT基因型频率分别为0.9%(1/110)、22.7%(25/110)、76.4%(84/110),C、T等位基因频率分别为12.3%(27/220)、87.7%(193/220);对照组CC、TC、TT基因型频率分别为0、9.0%(10/110)、90.9%(100/110),C、T等位基因频率分别为4.5%(10/220)、95.5%(210/220);两组间各基因型频率及等位基因频率分别比较,差异均有统计学意义(P<0.05)。2161-75C/T多态性位点基因频率、等位基因频率卵巢早衰组和对照组比较,差异均无统计学意义(P均>0.05)。结论本课题研究中建立的针对TGFBR3基因12外显子的变性高效液相色谱分析检测方法能够快速、准确地将TGFBR3基因12外显子变异序列与野生型序列区分,并且实验的重复性和再现性好、稳定性高,体系可靠。本研究建立的基于PCR的变性高效液相色谱分析TGFBR3基因12外显子突变方法是一种高通量、高灵敏度、半自动化、快速、准确、经济的突变检测技术。
Objective：To establish a rapid、accurate and economic method to detect TGFBR3 gene（exons 12）variations of the predisposing genes of POF. Methods：120 patients with idiopathic POF were recruited between February 2009 and December 2012 at the Affiliated Shenzhen City Maternity and Child Healthcare Hospital of Southern Medical University. Controls（n=110）were individuals under 40 with normal menstrual cycles,normal FSH concentrations. For the 12 exons and intron-exon junctions of TGFBR3 gene. primers were designed to establish DHPLC analysis assay. The 12 exons and intron-exon junctions of TGFBR3 gene were amplified in 2 reactions. Results：The denaturing high performance liquid chromatography analysis assay for TGFBR3gene（exons 12）can rapidly and accurately distinguish the sequence variation of known variations from wild-type sequence. This method based on PCR/DHPLC is a high-throughput,highly sensitive,automated,rapid,accurate and economical mutation detection method. Two nucleotide polymorphism（SNP）was identified in exon 12. For 2022 T/C polymorphism,Genotypic frequencies of CC、TC and TT（0.9%,22.7% and 76.4%）of POF group were significantly different from those of control group（0%,9.0% and 90.9% respectively,all P〈0.05）. Allelic frequency of C,T（12.3% and 87.7%）of POF group were significantly different from those of control group（4.5% and 95.5% respectively,all P〈0.05）. Allelic and genotypic frequencies of 2161-75C/T have not differed significantly between the two groups（P〈0.05）. Conclusions：Established for TGFBR3 gene in this study,the denaturing high performance liquid chromatography assay can distinguish the known variations of TGFBR3 gene from wild-type rapidly and exactly.This method based on PCR/DHPLC is a high-throughput,highly sensitive,automated,rapid,accurate and economical mutation detection method.
出处
《中国优生与遗传杂志》
2015年第11期32-34,共3页
Chinese Journal of Birth Health & Heredity
基金
深圳市科技计划项目(201202073)
深圳市科技研发资金(JCYJ2014041415370088)
深圳市卫计委科研项目(201401059)
