5-Aminoallyl-dUTP (5-[3-aminoallyl]-2'-deoxyuridine-5'-triphosphate) (cat. no. C401B) is supplied in lyophilized form. 5-Aminoallyl-dUTP is also available as a 10 mM solution in TE buffer. (cat. no. C401A).
5-Aminoallyl-dUTP can be enzymatically incorporated into DNA with Reverse Transcriptases, Taq DNA polymerase, phi29 DNA Polymerase, Klenow Fragment, Klenow Fragment, exo- and DNA Polymerase I. The resulting amine-containing DNA can be subsequently labeled with any amine-reactive fluorescent dye, biotin or hapten. This two-step method for labeling nucleic acids is considerably more economical than the one-step method using a prelabeled dUTP.
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Specifications:
Storage: Store at -20°C
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Applications
Enzymatic indirect non-radioactive labeling of DNA during cDNA synthesis, PCR, nick-translation, random-primed labeling, or primer extension.
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| Protocol (PDF): | C401B |
| MSDS (PDF): | MSDS-C401B |
Reference:
Science (2004) 305:846-846
Biotechniques (2000) 28:518-522
Nat Biotechnol (2002) 20:738-742
Frequently Asked Questions (FAQs)
Can dU-containing DNA produced by amplification with dUTP be cut by restriction enzymes?
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.
Why are there only two amino-modified nucleotides in the dNTP mix for cDNA indirect labeling (aminoallyl-dUTP and aminohexyl-dATP) intead of all four nucleotides being modified? Would more modified nucleotides have an adverse effect on the microarray?
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.
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