Aminoallyl nucleotide
Encyclopedia
Aminoallyl nucleotides are used in post-labeling of nucleic acid
s to be used in microarray
s. These nucleotides are formally known as 5-(3-aminoallyl
)-nucleotides since the aminoallyl group is usually attached to carbon 5 of the pyrimidine
ring of uracil
and cytosine
. They are usually abbreviated as aa-, such as aa-dUTP. The 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.
series, HiLyte Fluor
, or Alexa Fluor dyes that also been modified to be amine-reactive. A problem with this method is that the dyes react with the buffering agent
usually used for storage of nucleotides. Instead a carbonate
buffer is used.
The DNA
or RNA
from a sample is copied in vitro by T7 polymerase, RT polymerase or some other polymerase (except of eukaryotic origin) in a mix of nucleotides in which one, typically uracil (RNA and also DNA), is a mix aminoallyl-NTP and normal UTP. An alternative method is using thiol
reactive dyes.
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s to be used in microarray
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
s. These nucleotides are formally known as 5-(3-aminoallyl
Allyl
An allyl group is a substituent with the structural formula H2C=CH-CH2R, where R is the connection to the rest of the molecule. It is made up of a methylene , attached to a vinyl group . The name is derived from the Latin word for garlic, Allium sativum. Theodor Wertheim isolated an allyl...
)-nucleotides since the aminoallyl group is usually attached to carbon 5 of the pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
ring of uracil
Uracil
Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
and cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
. They are usually abbreviated as aa-, such as aa-dUTP. The 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.
Uses
The aminoallyl group in aminoallyl-modified nucleotides is reactive with dyes such as the cyanineCyanine
Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...
series, HiLyte Fluor
AnaSpec
AnaSpec Inc. is a biotechnology company headquartered in Fremont, California. Located in the Silicon Valley, it is a provider of custom and catalog research peptides, antibodies, dyes, assay kits, synthesis reagents, oligonucleotides, and PCR kits...
, or Alexa Fluor dyes that also been modified to be amine-reactive. A problem with this method is that the dyes react with the buffering agent
Buffering agent
A buffering agent is a weak acid or base used to maintain the acidity of a solution at a chosen value. The function of a buffering agent is to prevent a rapid change in pH when acids or bases are added to the solution. Buffering agents have variable properties—some are more soluble than others;...
usually used for storage of nucleotides. Instead a carbonate
Carbonate
In chemistry, a carbonate is a salt of carbonic acid, characterized by the presence of the carbonate ion, . The name may also mean an ester of carbonic acid, an organic compound containing the carbonate group C2....
buffer is used.
The DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
from a sample is copied in vitro by T7 polymerase, RT polymerase or some other polymerase (except of eukaryotic origin) in a mix of nucleotides in which one, typically uracil (RNA and also DNA), is a mix aminoallyl-NTP and normal UTP. An alternative method is using thiol
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
reactive dyes.