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Chapter 2 2.2 Results and Discussion In this chapter, PNA molecular beacons are designed in order to be used in the field of food science, in particular in connection with the authenticity of monocultivar extra virgin olive oil. In fact, the recognition of SNPs characteristic of a specific cultivar of olive can allow to certify a monocultivar product. The probes used here had been developed in a previous work 26 in order to recognize a specific cultivar of olive among twelve different cultivars. In particular, the cultivar taken in consideration was “Ogliarola Leccese”. For this cultivar, in fact, a thymine substitutes an adenine in the position 60 of the gene Actin. The PNA probes were synthesized in order to be complementary to the sequence containing the thymine, thus able to recognize this particular SNP. Table 2-1. PNA sequences and melting temperatures of the PNA:DNA antiparallel fullmatch/mismatch duplexes in phospate buffer (pH=7) at a 5µm concentration for each strand PNA Sequence Tm (°C) Tm (°C) ΔTm PNA/DNA PNA/DNA (°C) fullmatch mismatch Beacon 11 mer Fluo-Glu-ACTCATTCACC-Lys-Lys-(Dabcyl)-NH 2 53 36 17 Beacon 13 mer Fluo-Glu-TTACTCATTCACC-Lys-Lys-(Dabcyl)-NH 2 54 37 17 Beacon 15 mer Fluo-Glu-GGTTACTCATTCACC-Lys-Lys-(Dabcyl)-NH 2 65 51 14 PNA 13 mer H-TTACTCATTCACC-NH 2 54 35 19 The monomer in bold corresponds to the SNP identifying the cultivar “Ogliarola Leccese” Three stemless PNA beacons, as previously described 21 (Figure 2-4), were designed only differing for the length of the sequence: 11, 13 and 15mer; with the mismatch position in the middle. The scope was to study the effect of the length on the efficiency of the probe. The melting temperature values of the three PNAs with the complementary and mismatched DNA oligonucleotides are reported in Table 2-1 and compared with a 13-mer standard PNA with the same sequence. As expected, the selectivity decreased with the increase of the length. It is to be noted that, whereas the 11-mer and 13-mer PNA probes showed a certain similarity in 44
PNA Molecular Beacons the affinities towards oligonucleotides, the 15-mer higher stability values. PNA presented quite different and much Figure 2-5: Selectivity of PNA molecular beacons at different temperatures measured by fluorescence spectroscopy. Selectivity has been obtained calculating the ratio between emitted fluorescence at 522 nm of fullmatch and mismatch hybrid with excitation wavelength at 497nm. Discrimination properties were tested in solution by evaluating the fluorescence switching on in the presence of fullmatch and mismatch oligonucleotides for a 1µM solution in Tris buffer at pH= 8. The results shown in figure 2-5 were obtained by measuring the fluorescence emission at 522 nm for fullmatched and mismatched hybrids and calculating the ratio between the two values at different temperatures. In general, all probes show optimal discrimination above room temperature, suggesting that recognition experiments should be carried out in partially denaturing conditions. As expected, the optimumm temperature for the probe performance differs for the three PNAs according to the probe length: the longer the PNA, the higher the optimumm temperature. In particular, the 15-mer probe could discriminate the point mutation with reasonably good performances only at high temperature, confirming that this probe is not ideal for analytical purposes, while smaller probes seemed to be more efficient in the mismatch recognition especially at lower temperatures. Comparing the performances offered by the two shorter probes, it appeared that the 13 mer probe had a slightly better selectivity in the conditions of analysis. A drawback of the method emerged from the consideration that the probe in the closed form had a residual fluorescence that decreases the selectivity. A way to separate the aspecific 45
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Chapter 5 be printed, in order to m
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Chapter 5 Table 5-1. PNA sequences
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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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