An analysis of the folding of DNA three-way junctions in relation to a novel assay for DNA detection
An analysis of the folding of DNA three-way junctions in relation to a novel assay for DNA detection
Three-way DNA junctions (3WJ) are composed of three mutually complementary strands of DNA. The strands form three double stranded, helical arms which exchange at the branch point. These structures are important in recombination, slipped-strand DNA, RNA architecture and, more recently, for DNA detection in an isothermal assay (Signal Mediated Amplification of RNA Technology or SMART).
Understanding the dynamics of folding of DNA junctions, in particular the relationship between sequence and folding, allowed improvements to be made to the SMART assay. Sequences from the clinical setting of the SMART assay have been used to investigate three-way junction behaviour and to give an analysis of the folding of junctions in complex and simple systems.
By using gel retardation assays to look at the relative angles between arms in the junction, the effect of changing the sequence on the dominant conformation of the junction can be analysed. This technique has been used to show that sequences far from the junction branch point as well as those adjacent to it can have a large effect on the conformation of a 3WJ. Adding chemical linkers into the branch point can also have a dramatic effect, causing a junction to switch its dominant conformation despite no change in sequence. The extension of a primary within a 3WJ context has also been shown to be very dependent on the sequence and folding of a junction. This is thought to be due to the accessibility of the 3WJ branch point to DNA polymerase. This appears to be the major cause of a difference in signal strength produced in the SMART assay.
A novel technique was developed using redundant bases to generate every sequence combination around the branch point for a large-scale analysis. Junctions were separated by their mobility on a polyacrylamide gel into different conformers. The DNA in each band on the gel was purified, cloned and sequenced to give the identity of sequences which fall into each conformation. This analysis showed a number of sequences common to each band on the gel. When two sequences from different bands were synthesised, they separated well from each other and as predicted on a gel. They showed a strong tendency to correctly base pair even under rapid annealing conditions.
Melting studies were performed using the Roche LightCycler and junction stability was measured by a variety of different fluorescence methods. Placing the fluorophore and quencher on different pairs of arms provided the most useful melting data.
University of Southampton
Jones, Kerensa J
a263f57e-94c8-4b42-a49f-c04e1f8b8dec
2005
Jones, Kerensa J
a263f57e-94c8-4b42-a49f-c04e1f8b8dec
Jones, Kerensa J
(2005)
An analysis of the folding of DNA three-way junctions in relation to a novel assay for DNA detection.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Three-way DNA junctions (3WJ) are composed of three mutually complementary strands of DNA. The strands form three double stranded, helical arms which exchange at the branch point. These structures are important in recombination, slipped-strand DNA, RNA architecture and, more recently, for DNA detection in an isothermal assay (Signal Mediated Amplification of RNA Technology or SMART).
Understanding the dynamics of folding of DNA junctions, in particular the relationship between sequence and folding, allowed improvements to be made to the SMART assay. Sequences from the clinical setting of the SMART assay have been used to investigate three-way junction behaviour and to give an analysis of the folding of junctions in complex and simple systems.
By using gel retardation assays to look at the relative angles between arms in the junction, the effect of changing the sequence on the dominant conformation of the junction can be analysed. This technique has been used to show that sequences far from the junction branch point as well as those adjacent to it can have a large effect on the conformation of a 3WJ. Adding chemical linkers into the branch point can also have a dramatic effect, causing a junction to switch its dominant conformation despite no change in sequence. The extension of a primary within a 3WJ context has also been shown to be very dependent on the sequence and folding of a junction. This is thought to be due to the accessibility of the 3WJ branch point to DNA polymerase. This appears to be the major cause of a difference in signal strength produced in the SMART assay.
A novel technique was developed using redundant bases to generate every sequence combination around the branch point for a large-scale analysis. Junctions were separated by their mobility on a polyacrylamide gel into different conformers. The DNA in each band on the gel was purified, cloned and sequenced to give the identity of sequences which fall into each conformation. This analysis showed a number of sequences common to each band on the gel. When two sequences from different bands were synthesised, they separated well from each other and as predicted on a gel. They showed a strong tendency to correctly base pair even under rapid annealing conditions.
Melting studies were performed using the Roche LightCycler and junction stability was measured by a variety of different fluorescence methods. Placing the fluorophore and quencher on different pairs of arms provided the most useful melting data.
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Published date: 2005
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Local EPrints ID: 465825
URI: http://eprints.soton.ac.uk/id/eprint/465825
PURE UUID: 1a2b5593-75b6-4771-af1e-3513c0ac0d99
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Date deposited: 05 Jul 2022 03:13
Last modified: 16 Mar 2024 20:23
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Kerensa J Jones
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