The synthesis of modified nucleotide 5-triphosphates and their enzymatic incorporation into DNA
The synthesis of modified nucleotide 5-triphosphates and their enzymatic incorporation into DNA
The enzymatic non-radioactive labelling of oligonucleotides has become a valuable tool in applications such as oligonucleotide hybridisation probe synthesis and automated DNA sequencing.
A series of 2'-doxyuridine-5'-triphosphate analogues, possessing amine linkers at the C5 position of the base, have been synthesised for use in enzymatic labelling experiments. The amine functionality has been used to couple the 2,4-dinitrophenyl (DNP) group onto the nucleotide analogues. The enzymatic incorporation of nucleotide analogues into oligonucleotides by Taq DNA polymerase has been investigated using linear primer extension assays and PCR experiments. An increase in the ratio of nucleotide analogue to naturally occurring nucleotide (dTTP) present in reaction mixtures resulted in an increase in labelling density and a decrease in yield of labelled oligonucleotides. Good yields were obtained when replacing 50-90% of the natural nucleotide with the analogue. Kinetic studies showed that DNP labelled analogues were inserted with 5.1-5.9% the efficiency of dTTP.
The antibody-mediated detection of DNP labelled PCR products was carried out using HRP conjugated rabbit and anti DNP antibody. PCR products labelled with the amine linker-modified nucleotide NH2-11-dUTP were post-labelled by reaction with Oregon Green 488 N-hydroxysuccinimide ester. The fluorescent PCR product was used in in situ hybridisation experiments with good results.
University of Southampton
1999
Braven, Helen Theresa
(1999)
The synthesis of modified nucleotide 5-triphosphates and their enzymatic incorporation into DNA.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The enzymatic non-radioactive labelling of oligonucleotides has become a valuable tool in applications such as oligonucleotide hybridisation probe synthesis and automated DNA sequencing.
A series of 2'-doxyuridine-5'-triphosphate analogues, possessing amine linkers at the C5 position of the base, have been synthesised for use in enzymatic labelling experiments. The amine functionality has been used to couple the 2,4-dinitrophenyl (DNP) group onto the nucleotide analogues. The enzymatic incorporation of nucleotide analogues into oligonucleotides by Taq DNA polymerase has been investigated using linear primer extension assays and PCR experiments. An increase in the ratio of nucleotide analogue to naturally occurring nucleotide (dTTP) present in reaction mixtures resulted in an increase in labelling density and a decrease in yield of labelled oligonucleotides. Good yields were obtained when replacing 50-90% of the natural nucleotide with the analogue. Kinetic studies showed that DNP labelled analogues were inserted with 5.1-5.9% the efficiency of dTTP.
The antibody-mediated detection of DNP labelled PCR products was carried out using HRP conjugated rabbit and anti DNP antibody. PCR products labelled with the amine linker-modified nucleotide NH2-11-dUTP were post-labelled by reaction with Oregon Green 488 N-hydroxysuccinimide ester. The fluorescent PCR product was used in in situ hybridisation experiments with good results.
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Published date: 1999
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Local EPrints ID: 464001
URI: http://eprints.soton.ac.uk/id/eprint/464001
PURE UUID: 8bfec4a2-a634-4325-be1f-de91b6a560ab
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Date deposited: 04 Jul 2022 21:00
Last modified: 04 Jul 2022 21:00
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Author:
Helen Theresa Braven
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