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Title: The effect of modified nucleosides on DNA duplex and triplex stability
Author: Vadhia, Sunil Jayantilal
ISNI:       0000 0001 3541 6477
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2007
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To date, the single most effective method of improving base pairing affinity and binding of PCR primers, fluorescent probes and triplex forming oligonucleotides (TFO) ',h,';I,,+ destabilising mismatch base pairs has been the incorporation of modified nucleoside into these oligonucleotide structures. As a consequence, significant improvements have been made in the areas of human identity testing, forensic science analysis, pharmacogenetics/pharmacogenomics and anti-gene therapy. In an effort to improve the stability of these DNA duplexes and DNA triplexes further, we have synthesised and incorporated a series of cytosine, 7-deaza adenine, thymine and 3Hfuro-[ 2, 3-d] pyrimidin-2-one base analogues. By using a combination of UV melting analysis and fluorescence melting experiments, we have demonstrated that each of the base analogues gives a significantly higher base pairing affinity and binding selectivity when compared to their corresponding natural base. In addition, we have also incorporated these base analogues into PCR primers (7-deaza adenine) and fluorescent probe sequences (cytosine, 7-deaza adenine, thymine and 3H-furo-[2, 3-d] pyrimidin-2-one). Results from peR experiments show that the 7-deaza adenine base analogue does not adversely the functioning of Taq polymerase during amplification and therefore at the very least behaves similarly to adenine within a PCR primer sequence. In addition, all of the tLUlJre~jCel1tly labelled base analogues (cytosine, 7-deaza adenine, thymine and 3H-furo-[2, pyrimidin-2-one) show a significantly higher level ofbase pairing affinity and binding selectivity a complementary target sequence over a mismatched sequence.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available