CAJ | 학술논문

目的建立异柠檬酸脱氢酶1(isocitrate dehydrogenase 1,IDH1)基因突变的焦磷酸测序检测方法,确定该方法的检测灵敏度。分析焦磷酸测序法与直接测序法对于鉴定IDH1突变的差异。方法构建携带野生型和突变型IDH1基因的质粒,使用质粒优化焦磷酸测序方法。使用已知比例的野生型和突变型质粒作为模版,确定突变的检测灵敏度。针对96例胶质瘤患者手术切除标本的基因组DNA,分别使用直接测序法和焦磷酸测序法,鉴定IDH1基因突变类型,并比较。结果使用焦磷酸测序能够检测到低至2%的IDH1突变。直接测序检出突变阳性率为32.3%、焦磷酸测序检出突变阳性率为74.0%。结论焦磷酸测序法检测IDH1基因突变灵敏可靠,适合临床分子诊断。
목적건립이저몽산탈경매1(isocitrate dehydrogenase 1,IDH1)기인돌변적초린산측서검측방법,학정해방법적검측령민도。분석초린산측서법여직접측서법대우감정IDH1돌변적차이。방법구건휴대야생형화돌변형IDH1기인적질립,사용질립우화초린산측서방법。사용이지비례적야생형화돌변형질립작위모판,학정돌변적검측령민도。침대96례효질류환자수술절제표본적기인조DNA,분별사용직접측서법화초린산측서법,감정IDH1기인돌변류형,병비교。결과사용초린산측서능구검측도저지2%적IDH1돌변。직접측서검출돌변양성솔위32.3%、초린산측서검출돌변양성솔위74.0%。결론초린산측서법검측IDH1기인돌변령민가고,괄합림상분자진단。
Objective The present study aimed to establish a pyrosequencing method for IDH1 mutation examination, and quantify the sensitivity of this method. Furthermore, we tried to compare the differences between the direct sequencing and pyrosequencing for IDH1 mutation examination. Methods Plasmids carrying either wide-type or mutant IDH1 gene were constructed, and used to optimize the pyrosequencing method. The exact amount of mutant plasmids mixed with wide-type plasmids were served as templates for the pyrosequencing reaction to quantify the sensitivity of pyrosequencing based mutation examination. Both direct sequencing and pyroquencing methods were used to detect IDH1 mutations in 96 gliomas. Results Pyrosequencing detected as low as 2 % IDH1 mutation mixed in wide-type gene. Among the 96 glioma samples examined, 32. 3% of the samples were identified as carrying IDH1 mutations based on direct sequencing, while 74. 0% based on pyrosequencing method. Conclusion Pyrosequencing is a reliable and sensitive method in detecting IDH1 mutation, which is suitable for molecular diagnosis.