CONNECTING EPI-EDITING CARGO

Decades of research have given scientists a keen understanding of the GENETIC CODE, universal to all life. We know which DNA letters code for which amino acid sequences, how the sequence is copied, read and EXPRESSED, and which key enzymes are responsible for each step in these processes. As the GENETIC CODE is universal, bits of sequence from any organism can be RECOMBINED with one another to form new gene products, coding for potentially new protein functions. For example, the sequence for Green Fluorescent Protein (GFP) from jellyfish is routinely connected or FUSED to particular genes sequence. It must be inserted and ligated into place at the proper location, so that it is read for the correct amino acid fusion with the gene of interest. The end result is is a GFP connected gene, in which derived proteins glow green enabling tracking by researchers.  

RECOMBINANT DNA TECHNOLOGY has been around since the 1970's. Built on the original HOMING ENZYMES called RESTRICTION ENZYMES, it involves the site-specific cutting and resecting of select DNA fragments to generate new DNA products. 

The process is carried out in 3 steps, which are well animated in the video displayed below.

1. Amplification or synthesis of individual sequences of DNA that researchers wish to connect. They must be designed to overlap slightly.
2. Site specific digestion by RESTRICTION ENZYME of a delivery PLASMID or vector which will house the recombined DNA for copying in bacteria. 
3. Ligation of the recombinant DNA product into the delivery plasmid and delivered to (transformation) bacteria for copying.

The primary enzymes and biochemical steps responsible for each of the above processes (DNA amplication, digestion, ligation, and transformation) are well characterized and these reactions are generally conducted with purified enzymes and DNA in vitro. 

The recombinant DNA can subsequently be purified in large quantities and delivered to other cell types for study. In the case of EPIGENETIC ENGINEERING, this specifically involves using RECOMBINANT DNA TECHNOLOGY to generate custom coding sequences for new EPIGENETIC MODIFIERS. To methylate DNA for example, the sequence for "dead" Cas9 can be fused to that coding for DNA methyltransferase protein cargo.