) Of the estimated 100,000 individual genes encoded by the human genome, approximately 10-20% are expressed by the average cell. The expression of this subset of genes is a major determinant of a cell's properties. In addition to the control of cellular phenotype and the normal physiological processes of an cancer. Therefore understanding of the mechanisms of these normal and pathological processes requires organism. alterations in gene expression also underlies the etiology of diverse pathological processes such as identification. isolation and characterization of differentially expressed genes. Differential display (DD) technology was developed recently for this purpose and has emerged as the most commonly used method for the identification and cloning of differentially expressed genes. Despite of its wide use, the power of DD has not yet been fully realized due to several major technical and theoretical shortcomings. First, there has been a lack of systematic scheme for saturation screening in order to cover most of genes expressed in a cell and no attempt has been made for the automation of DD to ensure the reproducibility and high-throughput of the method. Secondly. it has been difficult to apply DD to systems where limited amount of samples from in vivo tissues are available. Third. there has been no efficient method for rapid and consistent recovery of the full length cDNA from the sequence information obtained by DD. without having to screen a library. Finally. no analogous method has been developed for differential display of the 5 termini of mRNAs. In this study. we propose to overcome these obstacles. (1) We provide theoretical basis and experimental support for the rational design of a set of SC arbitrary 13mers, which when used in combination with 3 one-base anchored primers will cover most of genes expressed in a cell. (2) We will adapt the Beckman Biomek 2000 robotic liquid dispensing workstation to automate the DD reaction preparations to ensure consistency and high throughput of such saturation DD screening. (3) A positive-selection cloning system will be combined with """"""""Reverse Northern"""""""" strategy to ensure rapid cataloging and screening of the cDNA probes generated by DD. (4) We plan to further increase the sensitivity of DD towards single cell level by optimizing primer designs, isotopes and PCR conditions. (5) Using oligo-capping technique, we will develop novel methodologies for the recover of 5' full length cDNA and differential display of 5 termini of mRNAs.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZCA1-RLB-Y (M1))
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Jacobson, James W
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Vanderbilt University Medical Center
Anatomy/Cell Biology
Schools of Medicine
United States
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