PCR基因芯片上荧光PCR反应的研究

目的探讨能否在基因芯片上进行不同的荧光掺入PCR反应并根据基因芯片上荧光的变化判断基因的变异.方法利用健康外周血,正常脐血DNA,X连锁遗传性铁粒幼细胞贫血(XLSA)家系成员6人外周血DNA做PCR-SSCP,并测序确定.设计检测此点突变的Taqman探针进行荧光PCR反应,在芯片上进行同样的反应,与上述反应结果进行对照.利用4步位点特异PCR结合SYBR荧光染料初筛HLAA2,并在芯片上进行同样的反应,与上述反应结果进行对照.结果PCR-SSCP分析与测序确定X连锁遗传性铁幼粒细胞贫血家系两患者的ALAS2基因第5外显子有G514A点突变,母亲、外祖母为杂合子携带者.设计Taqman探针时此家系中六位成员DNA与二份正常男性脐血DNA检测与预期结果相符.将上述结果中同样的样本移入芯片进行PCR反应,结果与常规荧光PCR反应的结果一致.SYBR荧光染料PCR反应与芯片上SYBR荧光染料PCR反应的结果与预期完全相符.结论利用TaqMan探针与SYBR Green荧光染料可以在PCR基因芯片上顺利进行PCR反应,根据基因芯片上荧光的变化检测出点突变与单核苷酸多态性.为基因芯片的临床应用打下了基础.

Experimental study of fluorescence PCR on PCR chips

Objectives: To prove if fluorescence PCR which is used for different purposes can be carried through successfully on PCR chips and whether the changes of fluorescence can be detected. Methods: A point mutation was identified by using SSCP and sequencing a hereditary X chromosome linked sideroblastic anemia family. A Taqman probe specifically to detect this point mutation was designed and used in fluorescence PCR. We carried out the fluorescence PCR on a gene chip and in tubes, and compared the results from the two methods. We also used the 4 steps of site-specific PCR and SYBR fluorescent staining to detect HLA-A2 on the chip and compared the results with that from routine method. Results: The point mutation of G514A in the exon 5 of ALAS2 in a hereditary X-linked sideroblastic anemia family was confirmed by PCR-SSCP and seqencing. The results of the 6 members in the anemic family and 2 normal male newborns using Taqman probe we designed and the fluorescence PCR on chip were the same as those form routine method. The results of the routine SYBR fluorescent staining PCR and the reactions on chip were consistent with the expectations. Conclusions: Taqman fluorescence PCR can be carried out successfully on the chip and is suitable for the detection of point mutation and single nucleic polymorphorim. Fluorescence PCR with SYBR fluorescence staining can be performed on the chip and may be useful for the screening of fusion genes in cancer samples.