Aminoallyl NTPs are used for indirect DNA labeling in PCR, nick translation, primer extensions and cDNA synthesis. These labeled NTPs are helpful because of their application in molecular biology labs where they do not have the capacity to handle radioactive material. For example, 5-(3-Aminoallyl)-Uridine(AA-UTPs) are more effective for high density labeling of DNA than pre-labeling the DNA. After the enzymatic addition of the NTPs, amine reactant fluorescent dyes can be added for detection of the DNA molecule. When incorporated into DNA or RNA molecules by DNA/RNA polymerase, 5-(3-aminoallyl)-UTP provide a reactive group for the addition of other chemical groups. Thus aminoallyl modified DNA or RNA can be labeled with any compound which has an amine-reactive group.
aa-NTPs incorporated into DNA/RNA in combination with a secondary dye coupling reagents can probe for an array analysis.
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.