功能基因组学.ppt

* Digest the double stranded cDNA with a sequence-specific restriction enzyme (an anchoring enzyme) that cleaves most transcripts at least once. Nla III is used as an anchoring enzyme since Nla III sites are known to occur approximately every 250 bp. 5. Cleave with Mme I, a Type IIS restriction enzyme (tagging enzyme). Mme I binds to a recognition sequence in the adapter adjacent to the CATG site and cleaves the cDNA ～21 bp downstream from the adapter, releasing a ~60-bp tag with a 2-bp overhang. The tag consists of ~40 bp of adapter sequence and ～21 bp of unique sequence from a single transcript. * 转录后基因沉默(PTGS)最早被认为只限于矮牵牛花和其它一些植物中，在最近几年, 才发现广泛存在于多种生物中，并在病毒抵抗和转座子沉默机制中发挥重要的作用。其中，由于RNA干扰引起的转录后基因沉默，作为一种可在多种有机体中敲除特定基因表达的工具，是最让人激动的发现之一，迅速成为分子生物学研究的热点之一。 * injected dsRNA — a mixture of both sense and antisense strands — into C. elegans (10). This injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Indeed, injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous genes expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in its first generation offspring (10). The potency of RNAi inspired Fire and Timmons to try feeding nematodes bacteria that had been engineered to express dsRNA homologous to the C. elegans unc-22 gene. Surprisingly, these worms developed an unc-22 null-like phenotype (11-13). Further work showed that soaking worms in dsRNA was also able to induce silencing (14). These strategies, whereby large numbers of nematodes are exposed to dsRNA, have enabled large-scale screens to select for RNAi-defective C. elegans mutants and have led to large numbers of gene knockout studies within this organism (15-18). Elbashir et al. (Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference i