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The study of cancer genetics has focused primarily, until now, on changes at the genomic level. However, activating, or inactivating, mutations/deletions/rearran ments in DNA are not the only way in which alterations in gene expression can occur. Since the development of subtractive hybridization, which screens for differentially expressed genes between related populations at the RNA level, evidence has accrued showing that alterations in downstream gene expression might play a significant role in the biology and evolution of cancer. Establishment of the novel technique of differential display (DD) 1,2(1,2) has facilitated studies in this new field that has been termed "expression genetics" 3). Compared with traditional methods of gene expression analysis DD possesses the advantages of simplicity, sensitivity, reproducibility, and speed. It is the current method of choice in expression genetics not only in the cancer biology field but also in such diverse areas as embryology, developmental biology, and neurobiology. This novel technique involves the reverse transcription (RT) of mRNAs with oligo-dT primers anchored to the beginning of the poly(A) tail, followed by a poly merase chain reaction (PCR) in the presence of a second arbitrary random primer. The amplified cDNAs of 3' termini of mRNAs obtained (with the same primers) from different samples can then be run side by side on a sequencing gel to allow differentially expressed genes, either upregulated or downregulated, to be visualized (see Fig. 1). By changing primer pair combinations approx 15,000 individual mRNA species from a mammalian cell may be analyzed reasonably quickly, thus providing a "fingerprint" of the mRNA in any particular cell type. Differential cDNA fragments can easily be recovered and reamplified from the gel and cloned into vectors for future screening and characterization work. The DD technique can be subdivided into five steps fig. 1. Principal procedures of differential display technique. |
