变性高效液相色谱检测视网膜母细胞瘤Rb1基因外显子

目的探讨变性高效液相色谱（denaturing high performance liquid chromatography，DHPLC）检测视网膜母细胞瘤（retinoblastoma，RB）Rb1基因外显子突变的情况。方法收集25例RB患者的肿瘤组织和3例健康者的外周血。提取其基因组，扩增Rb1基因的第3、7、9、12和26个外显子片段，再以DHPLC和DNA直接测序法分别检测其突变情况并进行比较。结果所有模板均扩增出5个外显子片段。DHPLC分析得第3、7、9、12和26个外显子的最优化检测柱温分别为54．4、55．2、53．4、53．4和55．2℃；第3、7、9和12个外显子中，25例患者和3例健康者均呈现同样峰形；而第26个外显子中，有2例患者呈现不同峰形。对所有片段进行测序，第3、7、9和12外显子的扩增片段中未发现突变。而外显子26的扩增片段中，DHPLC峰形和其他片段不同的2个样品也未出现突变。DHPLC和直接测序结果的差异无统计学意义（P〉0．05）。结论DHPLC能快速检测出Rb1基因外显子片段的突变位点，可以用于RB的检测。

Detection of exon mutation of Rb1 gene in retinoblastoma by using denaturing high performance liquid chromatography

Objective To investigate the exon mutation of Rbl gene in retinoblastoma by using denaturing high performance liquid chromatography（DHPLC）. Methods Twenty-five patients with retinoblastoma were performed eyeball enucleation. The human genomic DNAs were isolated from the tumor tissues of 25 patients with retinoblastoma and the peripheral venous blood of 3 healthy persons respectively. The 3^rd ,7^th ,9^th, 12^th and 26^th exon fragments were amplified. Then the exon mutation of Rbl gene was detected by DHPLC and direct sequencing. Results All 5 exon fragments were amplified in all templates. The optimizing column temperature of the 3^rd ,7^th ,9^th ,12^th and 26^th exon in DHPLC was 54.4, 55.2,53.4,53.4 and 55.2℃ respectively. The 3^rd 7^th ,9^th and 12^th exon of all templates showed the same peak form with DHPLC, while the 26^th exon in 2 patients showed a different peak form. By sequencing all the fragments, the mutation th th 12^th exon in 2 patients which could not be found in the 3^rd ,7^th and exon fragments. The same result was seen in the 26^th had a different peak form with DHPLC. There was no significant difference between the results got by the two different methods. Conclusions The exon mutation of the Rbl gene could be quickly detected by DHPLC,which could be used in the detection of retinoblastoma.