Exchangeable template reaction

DETAILED DESCRIPTION OF THE INVENTION Description of the Exchangeable Template Reaction (ETR) mechanism.
The ETR is a method for the synthesis of long polynucleotide DNA fragments using short synthetic oligonucleotides as templates for DNA polymerase.
The method is based on a cyclic mechanism involving three main components: (1) polymerase activity to synthesize double-stranded DNA, (2) enzymatic activity to create 3' terminal single-stranded regions, and (3) specifically designed synthetic deoxyoligonucleotides used as templates for the polymerase reaction.
The critical step is the enzymatic creation of a 3' terminal single-stranded region at the "growing point" of the synthesizing polynucleotide chain, which is used for the complementary binding of the next oligonucleotide as a template to continue the polymerase reaction.
The order of oligonucleotide additions for each cycle is encoded in each 3' terminal sequence.
At the 3' terminus of the growing DNA molecule a specific sequence of nucleotides can anneal with a complementary sequence of nucleotides from the synthetic oligonucleotide.
Thus, it is possible to synthesize a long DNA fragment in one step by simply combining the entire set of deoxyoligonucleotides in one reaction tube containing all the required enzymatic activities and incubating the mixture at the optimal temperature and optimal buffer.
Each cycle begins with the complementary binding of the 3' terminal region of a synthetic oligonucleotide with the 3' protruding region of double-stranded DNA (step 1 in FIG.
1).
After annealing, a DNA polymerase reaction occurs to create a second strand of DNA using the short synthetic oligonucleotide as a template for DNA polymerase (step 2 in FIG.
1).
After polymerization is complete, the double-stranded DNA has been extended by the length of the synthetic oligonucleotide.
To initiate the second round in the cycle of DNA synthesis, another enzymatic reaction occurs that creates a 3' protruding single-stranded region by removing several nucleotides from the 5' terminus leaving a 3' protrusion