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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 detect the exon 12 polymorphism of TGFBR3 gene by DHPLC technique. Methods From February 2009 to December 2012, 110 cases of premature ovarian failure diagnosed as obstetrics and gynecology in Shenzhen Maternal and Child Health Hospital Affiliated to Southern Medical University were premature ovarian failure group, menstruation regularity and sex hormone examination within the normal range of age-matched 110 women as control group. Denaturing high performance liquid chromatography (HPLC) was used to design the exon 12 of TGFBR3 gene by designing primers for denaturing high performance liquid chromatography (HPLC) based on the coding region and splice site of exon 12 of TGFBR3 gene. Results The established method of denaturing high performance liquid chromatography (LC-MS / MS) against exon 12 of TGFBR3 gene can distinguish known and wild-type sequences quickly and accurately with good sensitivity and stability. A total of 110 cases of idiopathic premature ovarian failure and 110 normal individuals were collected and detected by denaturing high performance liquid chromatography (HPLC) analysis of exon 12 of TGFBR3 gene. Two single nucleotide polymorphisms (SNP) .2022 T / C polymorphism: The frequencies of CC, TC and TT genotypes in premature ovarian failure group were 0.9% (1/110), 22.7% (25/110), 76.4% (84/110) T allele frequencies were 12.3% (27/220) and 87.7% (193/220) respectively. The frequencies of CC, TC and TT genotypes in control group were 0, 9.0% (10/110) and 90.9% 110). The frequencies of C and T alleles were 4.5% (10/220) and 95.5% (210/220) respectively. There was significant difference in genotype frequency and allele frequency between the two groups P <0.05) .2161-75C / T polymorphism locus gene frequency, allele frequency in premature ovarian failure group and control group, the difference was not statistically significant (P all> 0.05) .Conclusion The research established in this study The method of denaturing high performance liquid chromatography (HPLC) for the detection of exon 13 of TGFBR3 gene can differentiate the exon 12 of TGFBR3 gene from the wild type sequence rapidly and accurately, and has good repeatability and reproducibility, high stability, system By this study established based on DHPLC analysis TGFBR3 PCR gene exon 12 mutation method is a high throughput, high sensitivity, semi-automatic, fast, accurate and economical mutation detection techniques.