What We Do
Properties of DNA
Nucleic acids are information containing macromolecules in living matter consisting of several building blocks: sugar (ribose in the case of RNA; deoxyribose in the case of DNA), phosphate, and the four bases Adenine, Guanine, Cytosine, and Thymine (in RNA Thymine is replaced by Uracil). Sugar and phosphate form the backbone in which the A, C, G, T, or U bases are attached. The sequence of the bases is the essential code for biological information of the organism. Thus, for translation into an amino acid sequence of a protein the bases are grouped together into triplets called codons (there are 64 (43) possible triplets or codons). This genetic code is universal for all known living organisms. Its properties and tendencies are what drive the biotechnology industry today.
The first unique feature of nucleic acids is its ability to carry information. The second feature is to form specific antiparaliel heteroduplexes. This is based upon the possibility that Adenine can form a pair with Thymine (or Uracil in RNA) and Guanine can form a pair with Cytosine. The base pairing ability is the reason for identical replication, for information interchange and information transport, and for the identification of specific sequences by probing them with complementary nucleic acids.
Synthetic DNA and a few of its Applications
Some of the most traditionally important applications are probes for hybridization (for confirmation of the presence of a specific target sequence), primers for DNA sequencing and/or PCR, DNA or RNA for structural studies of molecules, and DNA fragments for site directed mutagenesis.
There are several key components essential to the chemistry of building an oligonucleotide on an automated synthesizer.
First, the starting monomer must contain one distinct coupling site. Other possible binding sites must be blocked efficiently by protecting groups. The starting the monomer is usually in the form of a solid support such as a succinyl CPG.
The first monomer to be coupled must be added. Like the start-monomer it must be effieciently blocked by protecting groups except for the coupling site. The site where the next monomer is going to be attached must of course also be protected and chemically active at the phosphorous site such as a phosphoramidite.
Coupling must be allowed under appropriate conditions and the newly synthesized dimer must be isolated and everything else must be removed.
The coupling site of the dimer to the next monomer must be deprotected without affecting any one of the other protecting groups.
Again the now deprotected dimer must be isolated, then the next monomer must be added and so on.
Once synthesis of the appropriate length is complete, all protecting groups must be cleaved off of the product and the product off of the support to yield biologically active DNA.
In automated synthesis these mentioned steps are broken down as follows: