Novel techniques for genetic analysis
Novel techniques for genetic analysis
Genetic analysis involves detection of nucleic acids in a sequence-specific manner. Typically, oligonucleotide probes labelled with fluorescent dyes are used to facilitate detection of their complementary sequences. The development of two fluorescent probe formats, each employing DNA-intercalators as a fluorescence quenchers is described. The first, in which the intercalator 9-amino-6-chloro-2-methoxyacridein is covalently linked to the probe, adjacent to a 5’-fluorophore, increases its fluorescence upon hybridisation to the target sequence, due to interaction of the quencher with the probe/target duplex. The hybridisation specificity of these probes has been demonstrated, culminating in their use in real time PCR. The second format, which involves binding of intercalators to the probe/target duplex from free solution, leads to a decrease in fluorescence upon hybridisation. A range of DNA-binding ligands have been screened for use in this context, leading to the use of ethidium bromide as an intercalating quencher in real time PCR.
Efficient hybridisation of labelled oligonucleotide probes to their complementary sequences in PCR products is important for sensitive PCR-based genetic analysis. This can be hindered by competition between the probe and one amplicon strand for the target sequence. Several solutions to this problem are evaluated. Among these, the use of 2’-deoxyinosine-5’-triphosphate (dITP) to produce amplicons with reduced Tm is outlined.
Synthetic oligonucleotide probes used for genetic analysis must be obtained in high purity. Two hydrophobic tagging monomers for synthesis of the probes used in the multiplex ligation dependent probe amplification (MLPA) assay have been developed. Introduction of the tags at the 5’-end of synthetic oligonucleotides > 100 nt in length facilitates their purification by RP HPLC.
University of Southampton
Ranasinghe, Rohan T
b29fc8b4-2a66-430a-85fa-ff1c9c261f32
2005
Ranasinghe, Rohan T
b29fc8b4-2a66-430a-85fa-ff1c9c261f32
Ranasinghe, Rohan T
(2005)
Novel techniques for genetic analysis.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Genetic analysis involves detection of nucleic acids in a sequence-specific manner. Typically, oligonucleotide probes labelled with fluorescent dyes are used to facilitate detection of their complementary sequences. The development of two fluorescent probe formats, each employing DNA-intercalators as a fluorescence quenchers is described. The first, in which the intercalator 9-amino-6-chloro-2-methoxyacridein is covalently linked to the probe, adjacent to a 5’-fluorophore, increases its fluorescence upon hybridisation to the target sequence, due to interaction of the quencher with the probe/target duplex. The hybridisation specificity of these probes has been demonstrated, culminating in their use in real time PCR. The second format, which involves binding of intercalators to the probe/target duplex from free solution, leads to a decrease in fluorescence upon hybridisation. A range of DNA-binding ligands have been screened for use in this context, leading to the use of ethidium bromide as an intercalating quencher in real time PCR.
Efficient hybridisation of labelled oligonucleotide probes to their complementary sequences in PCR products is important for sensitive PCR-based genetic analysis. This can be hindered by competition between the probe and one amplicon strand for the target sequence. Several solutions to this problem are evaluated. Among these, the use of 2’-deoxyinosine-5’-triphosphate (dITP) to produce amplicons with reduced Tm is outlined.
Synthetic oligonucleotide probes used for genetic analysis must be obtained in high purity. Two hydrophobic tagging monomers for synthesis of the probes used in the multiplex ligation dependent probe amplification (MLPA) assay have been developed. Introduction of the tags at the 5’-end of synthetic oligonucleotides > 100 nt in length facilitates their purification by RP HPLC.
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Published date: 2005
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Local EPrints ID: 465507
URI: http://eprints.soton.ac.uk/id/eprint/465507
PURE UUID: decb3b3c-6b93-4b43-a1d4-5e3f61a610d7
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Date deposited: 05 Jul 2022 01:30
Last modified: 16 Mar 2024 20:13
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Author:
Rohan T Ranasinghe
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