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In this study, we utilized modified nucleoside triphosphates with the aim of enhancing hybridization signal intensity across a wide range of probe sequences.

Modified ribo- and deoxyribonucleoside analogs have been used in a variety of applications [ 12 — 14 ], but their use has been limited in the field of microarrays [ 23 ]. We demonstrate that 2-aminoadenosine or 2,6-diaminopurine DAP , an analog of adenosine, significantly increases signal intensity for a wide range of probe sequences, whereas C-5 methylated pyrimidine analogs of cytosine and uridine decreased signal intensities across the entire range of probe sequences on our platform Figure 2.

Nucleoside Triphosphates and their Analogs: Chemistry, Biotechnology, and Biological Applications

Although incorporation of DAP did not uniformly increase the hybridization stability for all target-probe duplexes, even moderate incorporation of DAP DAP:A resulted in signal intensity increases up to fold for the majority of probes on our platform. More importantly, a significant number of probes that went previously undetected i. Utilization of such a modification may prove especially valuable for low abundant transcripts which may be difficult to quantify because of weak hybridization kinetics.

As shown in Figures 2B and 2C , the largest increases in signal intensities occurred for low and medium expressers relative to the unmodified control sample. It is apparent that the effect of DAP depends not only on the sequence compositions of target and probe, but also on the absolute abundance of specific transcripts in the target sample. It is of interest to note that the results we report here differ from those obtained by others [ 23 ]. Those studies reported increases in signal intensity for targets containing 5-methyl UTP while completely substituted targets containing DAP had the opposite or no effect.

Although we cannot completely account for the disparity of the results, one key difference in methodology between the studies is that their group was not able to normalize concentrations for modified and unmodified ATP targets because of dramatic differences in cRNA yield. Therefore, mass input of cRNA target samples were much greater for unmodified samples compared to the DAP-containing samples.

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Because the sample preparation used in this protocol is highly robust and reproducible [ 24 ], we were able to use equivalent mass inputs of all modified and unmodified targets during hybridization, allowing us to present our results quantitatively rather than qualitatively. However, we agree that complete substitution of DAP for adenosine may cause specificity to decline in certain sequence contexts, which may limit its usefulness in microarray applications.

However, we have found that reducing the proportion of DAP incorporated into target samples e. We have previously shown the dramatic impact of mixing on hybridization signal intensities [ 6 ]. Presumably, this impact is due to the effect of three dimensional diffusion of the target molecules and to the effect on how many probes reach equilibrium in the hybridization. It is possible that the degree of mixing during the hybridization can affect the number of probes which show a difference in intensity with substitution of DAP for adenosine as well as the magnitude of this difference.

Given these differences, it is not surprising that our results are not in line with those previously reported. These studies have shown that DAP substitution in oligonucleotide probes can increase thermodynamic affinities as reported directly by increases in T m and indirectly through increases in hybridization intensities. Modified nucleotides appear to drive the equilibrium particularly for low copy transcripts binding with their targets towards duplex formation.

Based on our experiments with extended hybridization times Figure 3 , use of DAP may prove to be a useful alternative to increase array sensitivity for studies that are unable to generate enough target to hybridize or are unable to increase hybridization times because of throughput constraints. Many studies have shown that a ribopurine rich strand bound to a deoxypyrimidine rich strand has a higher stability than the corresponding all deoxy strands which, in turn, have a higher stability than a deoxypurine rich strand bound to a ribopyrimidine rich strand [ 26 , 27 ].

Finally, earlier studies have noted that methylation of cytosines generating MeC in ribopolynucleotides stabilizes duplexes to the same extent as substitution of thymine MeU for uracil [ 28 ]. Thus, although it is still surprising that MeC and MeU decrease hybridization intensities, it is not surprising that the magnitude of the effects generated by MeC and MeU residues are similar. Earlier studies have shown that DAP, in DNA fragments, can widen the minor groove, as detected by reactivity towards uranyl nitrate or susceptibility to Dnase cleavage [ 29 ], and can relieve compressions in the minor groove associated with A-tracts [ 30 ].

In contrast, NMR and molecular modeling studies of an oligonucleotide duplex containing MeC have demonstrated that, while this duplex still has a B-DNA conformation, there are differences in the structural parameters and thermal stability relative to the unmodified duplex [ 31 ]. An earlier study also found, in two closely related octanucleotide duplexes, that although the methylated duplexes retained their B-DNA conformation, different structural and thermal stability effects were seen [ 32 ].

We have shown that while complete substitution of DAP for ATP may cause decreased base mismatch discrimination in certain sequence contexts, equimolar ratios of adenosine and DAP in cRNA target samples did not have significant detrimental effects on specificity Figure 4.

Remarkably, in one sequence context, the DAP modification enhanced specificity against mismatches. The use of DAP to increase hybridization signals is supported by the results that show equivalency of both ratio calls and ratio variability in DAP:A and control samples Figures 5 and 6. When all data points are considered in the differential expression ratio analyses, the two methods appear equivalent. Such increases are critical in order to sample as many genes as possible in any given microarray experiment.

For example, some investigators have analyzed only data for which the signal intensity is greater than approximately 0. Although this method minimizes the variance associated with the ratios, many genes and their expression changes are missed.

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Other platforms use a minimal intensity level to determine whether a gene is present or absent, prior to including this gene in expression analyses [ 35 ]. It is plausible that DAP modification, by increasing intensity levels, particularly for low expressors, could enable wider coverage in genome-wide expression analyses.

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

In summary, incorporation of modified nucleotides may not only lead to discovery and better quantitation of rare expressers in complex samples but also impact other aspects of the microarray experiment such as probe design prior to array fabrication and the statistical design of microarray experiments. The target preparation procedure generation of amplified cRNA or of first strand cDNA and the subsequent duplex formation may dictate the choice of which modified nucleotide to use, as we and others have found differential effects with various nucleotides.

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  6. We believe further investigation in this area could be fruitful. Commercially available 2-aminoadenosine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, and 5-methyluridine-5'-triphosphate TriLink Biotechnologies, Inc. This typically resulted in fragmented target with a size range between — bases. All probes on these microarrays are mer oligonucleotides spotted by piezoelectric technologies and covalently attached to a polymeric matrix [ 6 ]. These microarrays were hybridized, washed, and processed using a direct detection method of the biotin-containing transcripts by a Streptavidin-Alexa conjugate as previously described [ 6 ].

    Processed slides were scanned using an Axon GenePix Scanner with the laser set to nm, a PMT voltage of , and a scan resolution of 10 microns. Signal intensities for each spot were calculated by summation of the pixel intensities for each spot, followed by local background subtraction based on the median pixel intensity of the area surrounding each spot. Whole array data normalization, when used, was performed independently for each slide by dividing each spot's intensity after background subtraction by the median signal intensity of all test probes.

    All false positives, determined by visual inspection of the images, which were greater than 2-fold different between duplicate arrays were removed. The digested products were purified using Microcon YM-3 columns followed by centrifugation at g for 30—60 minutes.

    This solution was analyzed on an HPLC column equilibrated with 0. The concentration of the heterocycles was determined by the absorbance values at nm the wavelength where maximal absorption occurs for the heterocycles and the biotin-containing nucleoside concentrations were determined by the absorbance values at nm the wavelength where maximal absorption occurs for biotinylated cytosine and biotinylated uridine.

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    Quantitative measurements using thin-layer chromatography. J Biol Chem. Eur J Biochem. Nucleosides Nucleotides. Timofeev E, Mirzabekov A: Binding specificity and stability of duplexes formed by modified oligonucleotides with a hexanucleotide microarray. Genet Analy. J Mol Biol. EMBO Journal. An NMR and molecular modeling investigation. Nature Genetics. Download references.

    Correspondence to Abhijit Mazumder. AN performed the target preparations, microarray hybridizations, and data analyses, and drafted the manuscript. CZ performed the digestion and chromatography of the cRNAs. DD participated in design of the study and data analyses. AM conceived the study, participated in its design and coordination, and aided in drafting the manuscript. Reprints and Permissions.

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    Search all BMC articles Search. Abstract Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences.

    Describes as the "universal currency of energy," ATP is essential to cell function in all living things; nucleoside triphosphate was found to be essential to the assembly of DNA. Nucleoside triphosphates and their analogs; chemistry, biotechnology, and biological applications. Physiology and biochemistry section. Abstracts of Papers Presented at the th Meeting. Nucleoside triphosphates dntp for pcr kit mm 4 0. Cook, Chem. Schaffer, V. Robert, S. Bittner, S. Weinschenk, C. Ludwig, Acta Biochim.

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