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Global expression mapping of mammalian genomes

Meier-Ewert, Wolfgang Sebastian; (1994) Global expression mapping of mammalian genomes. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Global expression mapping of mammalian genomes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; cDNA libraries
URI: https://discovery.ucl.ac.uk/id/eprint/10097907
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