微量胎儿细胞中Y染色体锌指结构基因自动测序及影响因素的探讨

目的：在已建立的较简便、敏感的对性连锁遗传病胎儿进行产前性别鉴定方法的基础上探讨高温循环全自动测序中几个因素对测序鉴定ZFY基因结果的影响。方法：自行设计ZFY引物并建立了富集胎儿细胞结合巢式聚合酶链反应扩增男性特异性ZFY基因并进一步循环测序方法，计算机辅助设计的两对寡核苷酸引物位于Y染色体短臂的Ucdo43保守区内；同时对DNA纯度、浓度和测序过程中水的质量对DNA自动测序结果的影响进行回顾性分析。结果：实验表明利用孕妇外周血中微量胎儿细胞扩增男性特异性ZFY基因并进行高温循环全自动测序方法可行，结果可靠，在DNA纯度、浓度较低以及配制测序胶时所用纯水水质较差时，对测序曲线造成不同程度的影响，从而影响测序结果。结论：提示该方法检测胎儿DNA是可行的，同时在DNA自动测序鉴定产物特异性时需使用高纯度高浓度的DNA和高质量的纯水，要想获得较长的测序片段，所用模板浓度不能太低。

The study on the influence factors of ZFY gene automated DNA sequencing of fetal cell

Objective : To establish the method for prenatal sex diagnosis of the fetal cells from maternal blood carrying sex-linked genetic disorder and to investigate the influence of several factors on ZFY automated cycle sequencing. Methods:The oligonucleotied primers designed with computer were located at Y chromosome specific ZFY(zinc-finger-Y) gene. The effects of DNA purity and DNA concentration, as well as the quality of water used during automated cycle sequencing were retrospectively analyzed. Results:When the concentration ,purity and quality of water were not good ,they would lead to difficulty in correctly judging the sequencing. Conclusion:The results indicated that fetal sex determination by nest PCR was practicable.And high purity,concentration of DNA and high quality of water should be used in automated cycle seqeencing.