DNA Anchor/Terminator

Brick Creation

The use uracil-containing primers and USER^TM enzyme mix provides an alternative (and more effective) method for creating 12 base sticky ends. The primers anneal to the A and B regions respectively, as well as ~5bp 3' into the cassette (to increase melting temperature). Bricks cloned into pAB and pBA can be PCR'd up with these universal uracil primers prA1u/prB1u, prA2u/prB2u) and treated with USER^TM mix. The uracil DNA glycosylase (UDG) present will cleave the uracil base and endonuclease VIII will subsequently cleave the sugar-phosphate backbone at the apyrimidinic, creating single stranded regions which can be purified away using PCR purification spin columns. See Figure 4.

Figure 4: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions

Anchoring System

The new and improved method for brick production (ie: USER^TM) necessitated a change in anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER^TM-based anchoring system. An anchoring piece, constructed of two annealed oligomers, is bound to the streptavidin-coated bead via a 5' biotin modification and provides a sticky 3' overhang complementary to an A end. When the desired number of bricks is added, a terminator (again, two annealed oligomers) is annealed and ligated to the available end of the final brick (in this case, a B end). The entire construct is then treated with USER^TM enzyme mix. The resulting end, product from the digestion of uracil contained within the anchor, anneals to the terminator overhang and can be ligated to form a circular product. The ligation also yields a complete SceI site that can be used to linearize the construct for recombination into the E. coli genome. See Figure 5.

Figure 5: Showing anchor and terminator fragments and effect of USER^TM treatment. I SceI site and A ends are highlighted