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Studies on three-way DNA junctions related to the development of a novel method for the detection of genetic polymorphisms

Studies on three-way DNA junctions related to the development of a novel method for the detection of genetic polymorphisms
Studies on three-way DNA junctions related to the development of a novel method for the detection of genetic polymorphisms

Mutations in tlie genetic material of living organisms can have major effects on the survival of the individual. Genetic variations influence both the virulence and treatment-susceptibility of micro-organisms and affect the host-susceptibility for pathogenic micro-organisms. In addition, many common genetic disorders arise from defects in multiple alleles. This has important implications for health diagnostics as most available methodologies for genetic screening are not suitable to detect these. We have therefore developed a novel nucleic acid screening method, named signal mediated amplification of RNA technology (SMART), for detection of genetic polymorphisms. The technology uses two nucleic acid probes that anneal to a specific target sequence and to each other, resulting in the formation of a three- way DNA junction (TWJ). One probe (extension probe) can be extended against the remaining probe (template probe) by a DNA polymerase. This activity yields a functional T7 RNA polymerase promoter, and addition of T7 RNA polymerase results in RNA production. The RNA produced functions as a signal for the presence of a specific target sequence. These studies have focussed on optimising the junction design in SMART to allow discrimination between targets containing only small genetic variations. The results indicate that a number of factors can enhance the specificity of SMART: 1) incorporation of PNA (peptide nucleic acids) in the target complementary regions of the two probes, 2) shortening the size of the target containing duplex arms, 3) shortening the complementary sequence shared by the two probes, 4) elevating the reaction temperature. A further DNA-analogue (Locked Nucleic Acids, LNA) was also assessed but found to be less sensitive for mutation detection. The optimised assay is able to discriminate between the wild-type and single- and three-base variants of the cystic fibrosis transmembrane conductance regulator and Factor V genes, using either synthetic or PGR amplified genomic material. Additional studies also revealed that the structure of the TWJ may affect the RNA yield. In the presence of divalent cations, three-way junctions adopt two different coaxially stacked confonnations. The preferred conformer depends on the sequence around the branch point and this affects the efficiency of the assay. We have used a gel-electrophoresis method to probe the structures adapted by different junctions. The major factor influencing the conformer distribution is the arrangement of purines and pyrimidines around the junction, although some junctions appeared to adopt unusual conformations. Together these findings have improved the understanding of TWJ conformation and allow for more accurate structure prediction based only on the DNA sequence. Finally, a novel cleavage activity of restriction endonucleases was discovered and characterised. The activity was observed only for DNA constructs containing a non-DNA linker (e.g. hexaethylene glycol, abasic site) and involved cleavage of the phosphate located directly 5' of the linker. The cleavage was further found to be independent of the presence of canonical substrate and appeared to be modulated by methyltransferase treatment.

University of Southampton
Assenberg, René
7efbc447-f8d2-4cd5-9194-fc170989573a
Assenberg, René
7efbc447-f8d2-4cd5-9194-fc170989573a

Assenberg, René (2001) Studies on three-way DNA junctions related to the development of a novel method for the detection of genetic polymorphisms. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Mutations in tlie genetic material of living organisms can have major effects on the survival of the individual. Genetic variations influence both the virulence and treatment-susceptibility of micro-organisms and affect the host-susceptibility for pathogenic micro-organisms. In addition, many common genetic disorders arise from defects in multiple alleles. This has important implications for health diagnostics as most available methodologies for genetic screening are not suitable to detect these. We have therefore developed a novel nucleic acid screening method, named signal mediated amplification of RNA technology (SMART), for detection of genetic polymorphisms. The technology uses two nucleic acid probes that anneal to a specific target sequence and to each other, resulting in the formation of a three- way DNA junction (TWJ). One probe (extension probe) can be extended against the remaining probe (template probe) by a DNA polymerase. This activity yields a functional T7 RNA polymerase promoter, and addition of T7 RNA polymerase results in RNA production. The RNA produced functions as a signal for the presence of a specific target sequence. These studies have focussed on optimising the junction design in SMART to allow discrimination between targets containing only small genetic variations. The results indicate that a number of factors can enhance the specificity of SMART: 1) incorporation of PNA (peptide nucleic acids) in the target complementary regions of the two probes, 2) shortening the size of the target containing duplex arms, 3) shortening the complementary sequence shared by the two probes, 4) elevating the reaction temperature. A further DNA-analogue (Locked Nucleic Acids, LNA) was also assessed but found to be less sensitive for mutation detection. The optimised assay is able to discriminate between the wild-type and single- and three-base variants of the cystic fibrosis transmembrane conductance regulator and Factor V genes, using either synthetic or PGR amplified genomic material. Additional studies also revealed that the structure of the TWJ may affect the RNA yield. In the presence of divalent cations, three-way junctions adopt two different coaxially stacked confonnations. The preferred conformer depends on the sequence around the branch point and this affects the efficiency of the assay. We have used a gel-electrophoresis method to probe the structures adapted by different junctions. The major factor influencing the conformer distribution is the arrangement of purines and pyrimidines around the junction, although some junctions appeared to adopt unusual conformations. Together these findings have improved the understanding of TWJ conformation and allow for more accurate structure prediction based only on the DNA sequence. Finally, a novel cleavage activity of restriction endonucleases was discovered and characterised. The activity was observed only for DNA constructs containing a non-DNA linker (e.g. hexaethylene glycol, abasic site) and involved cleavage of the phosphate located directly 5' of the linker. The cleavage was further found to be independent of the presence of canonical substrate and appeared to be modulated by methyltransferase treatment.

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Published date: 2001

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Local EPrints ID: 464416
URI: http://eprints.soton.ac.uk/id/eprint/464416
PURE UUID: 1ce7ecd4-53c9-4b9f-94f5-dab38a15aebd

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Date deposited: 04 Jul 2022 23:36
Last modified: 05 Jul 2022 01:44

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Author: René Assenberg

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