A strategy for genome-wide gene analysis: Integrated procedure for gene identification

摘要

We have developed a technique called the Integrated Procedurefor Gene Identification that modifies and integrates parts from severalexisting techniques to increase the efficiency for genome-wide geneidentification. The procedure has the following features:(i) Only the 3′ portion of the expressed templates isused to ensure a match to 3′ expressed sequence tag (EST) sequences;(ii) the 3′ portion of the cDNA is poly dA/poly dTminus, which maintains complete representation of the expressed copies,particularly the rare copies, which otherwise would be lost heavilybecause of random poly dA/poly dT hybridization in the subtractionreaction; (iii) redundancy is decreased substantially bythe subtraction reaction to reduce the effort for sequencing analysis;(iv) the nonsubtracted templates that largely containthe rare copies are amplified selectively with suppression PCR and aresequenced directly or through serial analysis of gene expression(SAGE); and (v) the identified sequences are matched todatabases to determine whether they are cloned genes, ESTs, or novelsequences. Using this procedure in a model system, we showed that theredundant copies were largely removed, and the rates of EST matches andthe novel sequence identification were significantly increased. Most ofthe plasmids containing the matched EST are readily available from theIMAGE consortium. This technique can be used to index genome-wideexpressed genes and to identify differentially expressed genes indifferent cells. Compared with the existing techniques, this procedureis relatively efficient, simple, less expensive, and labor intensive.It is especially useful for standard molecular laboratories to performgenome-wide studies.