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Content archived on 2024-04-30

A new approach to CDNA finger printing and Development of a Commercial Kit for differential screening of gene transcription

Objective



A multitude of techniques has become available in recent times to isolate differentially expressed genes. Among these, random primed RNA fingerprinting is a powerful tool for the isolation of coding regions of transcriptionally regulated genes. The main objective of the proposed work programme is to evaluate the efficiency and the feasibility of a computer-assisted approach for searching a new set of RNA fingerprinting 12-mers (DNA primers) to develop a new protocol of cDNA fingerprinting for the differential screening of gene expression. The collection of reagents generated through this approach will allow us to propose a simple, exhaustive and systematic tool for the analysis of differential gene expression as a worktable, advantageous alternative to differential display techniques. The aim is to develop a marketable product for the differential screening of gene expression adapted to both manual operations and automatic workstations. The activity will concern theoretical PCR simulations performed on selected non-redundant databases to isolate a set of 12-mer oligonucleotides with particular thermodynamic parameters, the evaluation of the efficiency and specificity of the chosen primers by PCR amplification experiments on RNA extracted from tissues and the sequence analysis of cloned products. Preliminary studies, performed on a partial cDNA library, makes feasible the construction of different panels of 96 primers displaying marked affinity for coding portions of known genes and, presumably, of novel one, and useful for the development of micro-titer-plate commercial kits.

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