Product Code Database
Aminoallyl nucleotide is a nucleotide with a modified base containing an allylamine. They are used in post-labeling of by fluorescence detection in microarray. They are reactive with N-Hydroxysuccinimide ester group which helps attach a Fluorophore to the primary amino group on the nucleotide. These nucleotides are known as 5-(3-aminoallyl)-nucleotides since the aminoallyl group is usually attached to carbon 5 of the pyrimidine ring of uracil or cytosine. The primary amine group in the aminoallyl moiety is aliphatic and thus more reactive compared to the amine groups that are directly attached to the rings (aromatic) of the bases. Common names of aminoallyl nucleosides are initially abbreviated with aa- or AA- to indicate aminoallyl. The 5-carbon sugar is indicated with or without the lowercase "d" indicating deoxyribose if included or ribose if not. Finally the nitrogenous base and number of phosphates are indicated (i.e. aa-UTP = aminoallyl uridine triphosphate).
As instrumentation and technologies become more advanced in the field of DNA microarray, better reagents and techniques will be needed to further scientific studies. Fluorescent labeling with Cy3 was shown to be more insufficient and skew results; the method of aminoallyl nucleotide incorporation was opted instead. Using aminoallyl nucleotides as indirect fluorescent labeling seemed to nullify the sensitivity issues seen in cyanine-labeling.
In the image above, on the left is a modified nucleoside with an iodine (the iodine is added via electrophilic halogenation) in the fifth carbon in the pyrimidine ring. Its formation can be associated with a reaction with an allylamine and various reagents via heck coupling are able to remove the halogen group from the base and add the allylamine to become the aminoallyl nucleoside shown on the right. The product on the right is then used to in molecular biology in RNA synthesis.
Other reactions include using a single pot synthesis with other halogens.
cDNA relies on aminoallyl labeling for detection purposes. Although direct labeling of dNTP is the quickest and cheapest method of fluorescent labeling, it is disadvantageous as the sequence allows for only one modified nucleotide for use. Another disadvantage of direct labeling is the bulky nucleotides, however this can be overcome by indirect labeling using aminoallyl modified nucleotides. An easy way to check for labeling success is the color;Good labeling will result in visible blue (Cy5) or red (Cy3) color in the final material.
Another process which uses aminoallyl labeling is NASBA ( Nucleic Acid Sequence Based Amplification), a highly sensitive technique for amplifying RNA. In this specific case, the aaUTP modified RNAs were tagged with fluorescent market Cy3. NASBA combined with aminoallyl-UTP labeling is very useful for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology. DNA microarray is another method which utilizes specifically AA-NTP's making DNA microarray testing quicker and cheaply.
Post-synthesis labeling avoids the problems found in direct enzymatic incorporation of Cy-labeled dNTPs by generating probes with equal labeling effectiveness. With indirect labeling, amine-modified NTPs are incorporated during reverse transcription, RNA amplification, or PCR. Amino allyl-NTPs are incorporated with similar efficiency as unmodified NTPs during polymerization.
Concerns with labeling: The amine group, in aminoallyl-modified nucleotide, is reactive with dyes such as the cyanine series, or other patented dyes. A problem arises when the dyes react with buffering agents which are necessary for the proper storage of the nucleotides. However, a carbonate buffer can be used to overcome this problem.
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