![]() ( 6).ĭiagram showing how contaminating linkers are removed during the modification to the SAGE method. In order to assess directly the improvement that this modification gives, a side by side comparison was carried out between the method I describe below and an alternative protocol which aims to remove the contaminating linkers using a gel-purification step, described by Velculescu et al. I describe a modification to the SAGE method which efficiently removes linkers from the ditag concatenation reaction and generates clones with large inserts and high tag numbers. For SAGE to be efficient and to permit the analysis of large numbers of tags, the number of tags per clone must be as high as possible. This leads to fewer clones being generated which, in turn, contain very few tags per clone. The resulting molecule will not have compatible ends with which to clone into the prepared vector, pZero (Invitrogen). This unwanted linker ligation terminates concatenation of that molecule and the reaction is effectively poisoned. This serial analysis of many thousands of gene specific tags allows the simultaneous accumulation of information from genes expressed in the tissue of interest and gives rise to an expression profile of that tissue ( 1–5).ĭuring the generation of the ditags linker molecules persist, despite a gel purification step, which have compatible sticky ends enabling them to ligate to the ditags ( Fig. 1). ![]() The abundance of a particular tag relates directly to the expression level of the gene from which it is derived. Sequencing the clones allows over 30 individual tags to be read from each lane of an automated sequencing gel. The tags are generated as dimers, or ditags, and are ligated together to form concatemers which are then cloned. This allows specific detection of that cDNA from a large number of different transcripts. The Serial Analysis of Gene Expression (SAGE) method ( 1) generates short sequence tags which are positionally located within the cDNA molecule from which they are derived.
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