Vincent, John Bernard; (1994) Gene detection with synthetic oligonucleotide sequences. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes experiments to test the hypothesis that it is possible to detect coding regions in genomic DNA by using short synthetic oligonucleotides. Two types of sequences were targeted. The first consisted of sequences complementary to sites for rare-cutter restriction enzymes, which are often clustered in the CpG rich islands located adjacent to, or within, coding regions. The second were sequences with complementarity to consensus sites for control and regulation within or around coding regions, such as splice sites and transcription factor binding sites. Southern hybridization experiments were first carried out to test the hypothesis that short oligonucleotides, based on G/C rich regions and on consensus splice site sequences, could be used as hybridization probes to detect cosmids or fragments of cosmids containing CpG islands or splice site junctions. The cosmid vector pWE15 which contains two Notl sites and several clones containing the genes for human proteolipid protein, calcitonin/calcitonin gene related peptide-a (CGRP), glutathione-S-transferase and NADH-ubiquinone oxidoreductase were used as model systems for testing this hypothesis. Secondly, the polymerase chain reaction was used to test the hypothesis that short oligonucleotides based on rare cutter sites could be used as specific PCR primers using subclones of the cosmid vector pWE15 and phagemid pSL1180 as model systems, or in a less specific manner to amplify DNA in well characterised clones such as those containing the PLP, calcitonin/CGRP, glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes. In addition the hypothesis that this method could also be used on total human genomic DNA, so that clones enriched for either CpG islands or for coding regions could be obtained, using rare cutter site, splice site and transcription factor site oligonucleotides, was also tested. Several methods including a "TA" cloning strategy were employed in order to generate mini-libraries of the amplification product for subsequent evaluation. Degenerate oligonucleotides with just their 3' ends based on the splice site and translation start site consensus sequences and with cloning sites at their 5' ends were also used on the model clones to test whether coding regions could be identified. Thirdly, experiments were carried out to demonstrate that short oligonucleotides based on rare cutter sites could be used as PCR primers for sequencing directly into CpG islands/coding regions in cloned DNA. Subclones of the four genes mentioned above were used as models to test this. Finally, experiments were carried out to test the hypothesis that the ligation of primer/linkers to rare-cutter restricted cosmids followed by direct PCR-sequencing could be used to obtain direct sequence from putative CpG islands in cloned genes. Several cosmid clones including the glutathione-S-transferase and NADH-ubiquinone oxidoreductase genes were used for this. The experiments showed that some of the hypotheses concerning G/C rich sequence detection could be confirmed. When genomic DNA was used as template, G/C rich oligonucleotides as short as 8-mers could prime PCR amplification and enable "TA" clones to be produced which were enriched 66-fold for CpG rich sequences. In addition, conditions necessary for direct and specific amplification using G/C rich oligonucleotides as short as 7-mers with vector constructs as model target DNA were identified. However, PCR using G/C rich oligonucleotides was not capable of identifying CpG islands within cosmid clones. G/C rich 8-mer oligonucleotides may also be used in certain situations for directed sequencing within cloned genes and could thus be used as random or directed primers in a large volume sequencing project. Primer/linkers for rare-cutter restriction sites may also be used for sequencing into CpG islands within cosmids. Oligonucleotides that have their 3' ends complementary to the splice site consensus sequence can also prime amplification (Degenerate Oligonucleotide Primed-PCR) from some splice sites in some cloned genes, although with low success rate. In conclusion, the development of methods to enrich for CpG islands in genomic DNA was successful, but identifying CpG islands in cosmid clones was not. However a degree of success was achieved in the direct sequencing of CpG islands within clones. The work with splice site sequences was less successful, and it must be concluded that other methods should be employed for the identification of coding regions within cloned DNA.

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