Hampson I N, Hampson L, Dexter T M
Department of Experimental Haematology, Paterson Institute of Cancer Research, Christie Hospital, Manchester, UK.
Nucleic Acids Res. 1996 Dec 1;24(23):4832-5. doi: 10.1093/nar/24.23.4832.
We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.
我们描述了一种对cDNA进行全基因组PCR扩增并保持链方向的方法。该过程的产物是大小在100至500 bp之间的随机引物片段，这有助于对总cDNA进行均匀的PCR扩增。T7 RNA聚合酶起始子/启动子序列的定向掺入允许从该材料高效合成有义总RNA，并且使用生物素化引物可实现单链cDNA的分离。从不同细胞类型中分离这些产物可从有限数量的细胞中提供可再生的靶单链cDNA和驱动RNA来源，并且我们展示了它们在差异表达cDNA的消减杂交克隆中的应用。
