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Nevertheless, it appeared quite clear that the strongest amplification signals belonged to tomato leaves and fruits, due to the better quality of extracted DNA compared to the processed one. Indeed, in the lanes corresponding to tomato derivatives a lower band can be observed, due to the primers and dNTPs which were not utilized in the reaction efficiently, because of the scarce availability of the template substrate. The same band was also observed in the water negative control, where no amplification occurred at all. Albeit increased, the concentration of primer pairs targeting the locus LeOH 31.3 didn’t improve the amplification efficiency, as shown by the weak band corresponding to the LeOH 31.3 amplicon, whose visualization was further made worse by the slight difference in bp with the LeOH 23.1 amplicon (130 bp and 118 bp, respectively). Although the reduced efficiency of a primer pair, the multiplex PCR procedure here proposed appeared to be a valid method for the simultaneous amplification of the four selected tomato loci, laying the foundations for following hybridization on PNA microarray.
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DNA strand in a double-stranded DNA. The resulting Cy5-labeled single stranded target amplicon was then used for the hybridization on the array carrying both PNA 1 and PNA 4. Unfortunately, no specific hybridization signal was still obtained.
Different purification steps were then added to the previous PCR systems, in order to remove any possible contaminants, such as polyphenols derived from the DNA extraction step, or excess of primers and dNTPs, which could interfere in the PNA-DNA duplex formation. For this reason all the described PCR experiments were followed by a purification step, performed by EUROGOLD Cycle-Pure Kit protocol, and the residual DNA amount was quantified by Qubit® fluorometer, in order to assure that amplified fragments to be hybridised were still present in solution after purification. Neither this modification allowed us to obtain an improvement in the performance of DNA-PNA duplex formation on array.
Since in all the described hybridization experiments the positive control, that is the PNA on array hybridized with the short oligonucleotide mimicking the complementary genomic sequence, always gave a signal indicating a specific PNA–
DNA complexation, we wondered if the absence of any specific signal when using PCR products could be ascribed to a non-properly working amplification reaction or to a problem related to the length of the sequence to be recognized. For this reason an investigation of the microarray performance with long synthetic DNA sequences, mimicking amplified DNA sequences extracted from real food samples, was undertaken.
In particular, the LeOH 23.1 sequence was chosen for testing hybridizations with long DNA tracts. In order to study if and how the different positions of the target sequence in the eventual amplicon would affect the DNA detection, different hybridization experiments by using longer DNA oligonucleotides containing the target sequences in the middle, at the 5’ and at the 3’ ends, were performed. The different sequences used for hybridization experiments were three 118 bp oligonucleotides differing in the position of the target sequence inside the oligomer, and one shorter variant of one of them (60 bp) (Fig 6).
Fig 6. Design of DNA oligonucleotides mimicking the amplified DNA region at the locus LeOH 23.1. The bold red letters indicate the region to be recognized by PNA 2.
Hybridization experiments with the four DNA single strand sequences were performed under the same conditions used for short oligonucleotides. In all cases, no signals indicating specific PNA–DNA complexation were observed, with or without a preliminary incubation step at 95 °C for 5’ and/or the hybridization performed at room temperature and at 40 °C. The use of Tween 20 replacing SDS as a non-charged surfactant did not allow us to observe improved results, as well as the use of denaturating conditions (with the addition of small percentages of formamide), in order to make the PCR product more accessible to the PNA probes.
Thus these results seemed to indicate that the chiral box PNA microarray platform, albeit very specific, looses binding efficiency in the presence of longer DNA
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sequences, preventing the formation of PNA–DNA duplexes, and this problem, rather than the possibility of non efficient PCR systems, was the cause of the lack of detection obtained with amplicons. Although in previous work it had been shown that on DNA microarray devices the distance between the fluorophore, used to label hybridization, and the target sequence affects the efficiency of detection,22 in our case this phenomenon seemed not to be responsible for this behaviour, since the target sequence in our experiments had been designed at different distances from the fluorophore (Fig 6).
It may be speculated that the nature of the solid support and the presence of positive charges on PNA probes are more likely responsible for this effect, since they could promote the aggregation of the PNA on the surface, lowering the efficiency of the hybridization. Alternatively, long DNA tracts may aggregate on the positively charged surface, making the target DNA difficult to be bound by the PNA probes, and thus easily rinsed away during the washing steps.
Whatever might be the cause, it has already been demonstrated in the previous chapter that chiral PNA microarrays give less intense signals when hybridized to complementary oligonucleotides, as compared to standard PNAs, showing a higher specificity but a lower sensitivity in DNA detection. Given the promising potentialities of this kind of probe, future studies will have to be implemented in order to better understand the kind of interactions between the positively charged PNA probes, the microarray surface and long DNA tracts, which lowers the PNA binding efficiency.
5. Conclusions
The Arg-PNAs designed and synthesized in this work, tested both in solution and on microarray systems, confirmed the ability to perfectly discriminate sequences containing SNPs and thus their potentiality to be used to genotype tomato varieties by microarray approach. The microarray system here presented was able to simultaneously discriminate tomato varieties, except for ‘Talent’ and ‘Tomito’, in simulation experiments using oligonucleotide mixtures. Although the efficiency of DNA binding was somewhat lower than that usually shown by standard PNA arrays, causing a lower sensitivity towards long DNA sequences in this array system, the unsurpassed specificity of the PNA probes makes them very promising for the development of PNA-based genotyping methodologies. Further studies will be needed in order to better clarify the interactions occurring during positively charged PNA probes-long DNA tracts duplex formation on microarray surface in order to develop modified PNA-based systems able to compete in the future with the commercially available oligonucleotide-based recognition systems.
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