GLOBAL COMPETITIVE PCR FOR STUDYING GENE EXPRESSION

描述：（申请人描述）本项目的长期目标

DESCRIPTION: (Applicant's Description) The long term objective of this research is to study changes in global, i.e. genome-wide gene expression patterns in human tissues associated with cancer, aging, and exposure to environmental stresses. The specifics of studies in human tissues are that they require the ability to process a large number of samples and to extract information from small amounts of starting material. Existing approaches to measure gene expressions either concentrate on a few target genes or are relatively laborious, or/and require substantial amount of sample. There is an urgent need for a procedure that would amplify entire mRNA pools from small starting amounts and would be compatible with such labor-efficient but mRNA-consuming approaches as hybridization to DNA arrays. The problem is that during amplification of a complex mixture of sequences such as an mRNA pool the abundances of individual sequences may be unpredictably biased. We propose to develop a "global competitive PCR" procedure that will amplify the entire mRNA pool extracted from the samples to be compared combined in a single reaction tube. The approach is based on the attachment of a sample-specific tagging sequence and a pair of common primers to each mRNA sequence in the pools to be amplified. As in competitive PCR, ratios between identical (except for the tag) templates originating from different samples and thus the information about relative gene expression levels, is preserved during amplification. After amplification, the sequences originating from different samples are differentially labeled according to the tags they bear and are hybridized to a DNA array to measure, for each gene, the ratio of its expression levels in the two samples. The ratios can further be converted into absolute levels of expression. Initial experiments on a model system involving a set of bacterial DNA sequences of different length demonstrated the feasibility of the key steps of the proposed procedure.