实时荧光定量PCR技术在检测外源基因拷贝数中的应用

通过对PCR法、Southern Blot法、IPCR法与实时荧光定量法4种转基因外源基因检测方法的比较认为:PCR扩增虽十分灵敏,但有时会出现假阳性扩增,因而对外源基因是否整合还需进行验证.Southren方法准确性高,特异性强,但存在费时、费力的缺点.同时实际操作中就需要较大量的植物材料来提取DNA,而转基因植物的愈伤组织在无菌条件下经过筛选、重新分化后,一般都比较细弱,不宜大量取样.IPCR法虽可以转座突变分离基因,但当转座子作为外源基因通过农杆菌介导等方法导入植物时,由于T DNA整合到染色体中引起插入突变并分离基因造成误差.实时定量PCR技术的特异性和高信噪比为转基因拷贝数定量提供了方便,实时定量PCR法,花费试剂少、节省劳力和时间,需要DNA样品量少、并不进行放射检测.同时对实时荧光定量PCR法计算进行了详细介绍.

The Application of Quantitative Real-time Fluorescent PCR Techniques in Detecting the Copy Number of Transgenic Gene

Comparison on the methods for detecting transgene in transgenic plants were conducted in this paper, including Polymerase chain reaction (PCR), Southern Hybridization, Inverse PCR ( I-PCR) as Real time PCR with flurosense-labelled primers. The conclution was as the followed: PCR was sensitive, but with some false positive results and not detecting the copy number; Southern hybridization was with higher acutivity and speciality, but with much more labor and time consumptio and also not detecting copy number; I-PCR was effective but also with some false positive, especially used for detecting copy numbers of transgene, and the Real time PCR could detect the transgene as the copy number as well with higher speciality and acurity. Therefore Real-time PCR with flurocence labelled primers could be used in the detection of the transgene and the copy number as well with the advantages of simpleness, quickness, credibility and short needs of target fragment DNA.