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Drs. Peng Liang and Arthur B. Pardee assemble for the first time a comprehensive review of the state of the art of their powerful new methodology and practical applications. The book's pioneering contributors describe all the major elements of this novel technology, including both RAP-PCR and DD using fluorescence detection, as well as their powerful strategy for identifying and cloning family-specific genes. They also provide numerous examples in which differentially expressed genes were successfully identified in diverse biological systems ranging from plants to songbirds to humans.
E-Book Content
1 Differential
Display
A General Protocol Peng Liang and Arthur B. Pardee 1. Introduction One of the greatest unsolved mysteries of life 1show the hundreds of thousands of genes embedded in the genome of an organism are selectively expressed mto the mRNA and protems m a temporally and spatially regulated manner that gives rise to different tissues and organs. The abnormality m this intricate regulatory cn-cuitry IS beheved to be one of the underlmmg causes of a variety of pathological alterations or disease states.The rsolation and characterization of differentially expressed genes becomes one of the first steps toward the understanding of these important biological questions. Differential display (1) and a related RAP-PCR method (2) were developed to more efficiently Identify and isolate these genes. The general strategy for differential display (Fig. 1) IS based on a combmatton of three techniques brought together by a concept: 1 Reverse transcrlptlon of mRNA from anchored primers (see Note l), 2 Choice of arbitrary primers for setting lengths of cDNAs to be amplified by the polymerase chain reaction (PCR), each corresponding to part of a mRNA (tags), 3 Sequencmg gels for high resolution of amplified cDNA
The objective IS to obtain a tag of a few hun