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Chapter 2 lower sensitivity, and significant peaks could be obtained only by using relatively higher concentrations (200nM, Figure 2-7). Finally, although some good chromatograms were obtained, a high variability in the peak intensity was noticed, making the probe not particularly suitable for this assay. To make bad things worse, a PNA-DNA hybrid could be clearly detected also when the beacon was hybridized with the mismatched oligonucleotides. Figure 2-7: Chromatogram obtained for fullmatched and mismatched hybrids using PNA Molecular Beacon 15 mer 200nM at different temperatures. The analytical conditions are the same, as described above Since the only difference between this PNA and the other previously reported is the length, these problems should be related to this parameter. The increased length may cause selfaggregation in solution, due to the increased hydrophobicity, and a slower rate in the hybridization process also with complementary sequences. Moreover, the 15-mer beacon is less selective on account of the higher stability of the fullmatch and mismatch PNA-DNA hybrids at room temperature. In order to completely denaturate the mismatched duplex, the column temperature was increased. The chromatograms in figure 2-7 show that warming up to 35°C was necessary in order to get rid of the aspecific signal, but at this temperature the fullmatch hybrid was also highly destabilized, as demonstrated by its strongly decreased signal. Since all the results suggested that the optimal probe, in terms of selectivity and sensitivity, was a PNA beacon 13 bases long, this probe was used in the optimized conditions for the recognition of the DNA samples extracted from olive leaves and amplified by PCR. DNA extracted from leaves of two different cultivars of olive were used: one from “Ogliarola Leccese”, bearing the SNP at position 60 (thymine versus adenine) of the actine gene, for which the probe has been designed; the other from “Canino”, which was chosen as negative control, since it has an adenine at position 60 as other eleven cultivars. The extracted DNA was amplified by Polymerase Chain Reaction (PCR) and purified with an affinity column. 48
PNA Molecular Beacons The sample thus obtained was hybridizedd with the 13 mer PNA beacon 100nM and analyzed by AE-HPLC using the same conditions optimized with synthetic oligonucleotides. Figure 2-8: Chromatograms obtained hybridizing amplified DNA PNA Beacon 13 mer extracted from olive leaves with The chromatograms obtained (Figure 2-8) show a very good recognition of the complementary DNA. The DNA extracted from leaves coming from “ Ogliarola Leccese” gave a clear peak, whereas the other sample, containing the DNA extracted from leaves coming from “Canino”, with a mismatched base, didd not give any signal, confirming the good performances of this PNA beacon-based HPLC methodology. 2.3 Conclusions In this work an extensive study has been done on PNA molecular beacon performances for SNP detection of oligonucleotides and PCR products of DNA extracted from olive plants. On the basis of the results obtained it is evident that the design of the PNA beacon is a crucial point, and in particular the length is an important factor for its performance. Binding affinity and selectivity assays demonstrate that in this case a 13mer probe showed the best performances. The probe was applied to the identification of SNPs in real sample allowing a good discrimination of DNA from different olive cultivars. The method here developed can be considered a fast, robust and sensitive assay for DNA analysis, which does not require DNA labeling and is performed on a commonly available instrument (HPLC). 49
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Università degli studi di Parma Do
Page 3 and 4: 2.4.4 Fluorescence measurements ...
Page 5: Chapter 6 The double nature of Pept
Page 8 and 9: Preface In Chapter 2 a particular t
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Page 27 and 28: PNAs: from birth to adulthood amino
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Page 45 and 46: Chapter 2 Label-free detection of S
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Page 63 and 64: Arginine-PNAs monomers in the middl
Page 65 and 66: Arginine-PNAs The synthesis of the
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Page 71 and 72: Arginine-PNAs chain + Boc methyl gr
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Page 81 and 82: Arg-PNA Microarrays a surface. Alth
Page 83 and 84: Arg-PNA Microarrays 4.2 Results and
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Page 87 and 88: Arg-PNA Microarrays PNA-microarray
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Page 91 and 92: Arg-PNA Microarrays complex mixture
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Page 95: Arg-PNA Microarrays 30 T. Tedeschi,
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Chapter 5 printing the PNA on a gla
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Chapter 5 22.4°C 24.0°C 25.5°C 2
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Chapter 5 PNA 3 PNA 5 PNA 6 Slide 1
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Chapter 5 times. The substrates wer
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Chapter 5 chemicals were used as re
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Chapter 5 5.4.6 Microcontact Printi
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Chapter 5 5.5 References 1 A. L. Be
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Chapter 6 6.1 Introduction The use
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Chapter 6 Among all the peptides us
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Chapter 6 Figure 6-2: A standard PN
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Chapter 6 peptide 1 and the PNA tri
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Chapter 6 6.2.2 Uptake experiments
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Chapter 6 (HBTU), N-[(dimethylamino
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Chapter 6 1.41 (s, 9H, Boc methyl g
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Chapter 6 Arg + CHα Lys), 4.0-4.6
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Chapter 6 12 B. P. Gangamani, V. A.
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Contributions Publications includin
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