5-(3-Aminoallyl)-dUTP (50 mM)

Using four modified nucleotides rather than two can cause fluorescence quenching since the fluorophores are positioned very closely to one another. Use of two amino-modified nucleotides in the cDNA synthesis reaction (rather than one) results in a greater incorporation of fluorescent dye and higher signal intensity with small amounts of RNA starting material. Unbiased incorporation of amino-modified dNTPs and the high efficiency of the coupling reaction result in an even distribution of fluorescent signal and high overall levels of fluorescence, increasing the sensitivity and reproducibility of array hybridizations. In addition to quenching, a very high dye density on the cDNA will interfere with hybridization and, therefore, yield lower microarray fluorescence signals. We found that the use of 2 amino-modified nucleotides (at the appropriate concentration) results in optimal incorporation and high microarray signals.

You will need to confirm with the manufacturer of the restriction enzyme you are using to find out whether or not it can recognize dUTP-containing DNA.

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