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目的 建立一个适用于大规模基因突变扫描的荧光标记单链构象长度多态性检测方法。方法 共 2 7个位于肝核因子 4 α基因 ,葡萄糖激酶基因和肝核因子 1 α基因的已知突变位点用于检测 ,其中包括2 2个点突变、3个插入突变 (分别是插入 1、2和 7个碱基对 )以及 2个缺失突变。采用巢式PCR扩增目标片段并用绿色、蓝色和黄色三种荧光标记物标记。荧光测序仪电泳检测 (含 5 %甘油或 10 %蔗糖的非变性凝胶电泳 )。结果 结合两种电泳条件 93% (2 5 /2 7)的突变可被检出 ,而单用 5 %甘油或 10 %蔗糖的非变性凝胶电泳的检出率分别是 82 % (2 2 /2 7)和 6 7% (18/2 7)。结论 荧光标记单链构象长度多态性检测方法敏感、无污染、高效、省时 ,可用于大规模基因突变扫描
OBJECTIVE: To establish a fluorescent-labeled single-strand conformation length polymorphism detection method suitable for large-scale gene mutation scanning. Methods A total of 27 known mutations located in the hepatic nuclear factor 4 α gene, glucokinase gene and hepatic nuclear factor 1 α gene were used for the detection, including 2 2 point mutations and 3 insertions (insert 1 , 2 and 7 base pairs) and 2 deletion mutations. The target fragment was amplified by nested PCR and labeled with three fluorescent markers, green, blue and yellow. Fluorescence sequencer electrophoresis (non-denaturing gel electrophoresis with 5% glycerol or 10% sucrose). Results 93% (25/27) of the mutations combined with the two electrophoresis conditions were detected, while the detection rates of non-denaturing gel electrophoresis with 5% glycerol or 10% sucrose were 82% (2 2 / 2 7) and 67% (18/2 7). Conclusion The detection method of fluorescence single-strand conformation length polymorphism is sensitive, non-polluting, efficient and time-saving, and can be used for large-scale gene mutation scanning