Team:Alberta/DNAanchor

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University of Alberta - BioBytes










































































































DNA Anchor/Terminator

Brick Creation

The use uracil-containing primers and USERTM enzyme mix provides an alternative method for creating 12 base sticky ends that is more versatile than conventional restriction endonucleases. 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 the universal uracil primers prA1u/prB1u (for pAB), or prA2u/prB2u(for pBA) and treated with USERTM mix. The uracil DNA glycosylase (UDG) present in the mix will cleave the uracil base off the DNA backbone. Endonuclease VIII will subsequently cleave the sugar-phosphate backbone at the apyrimidinic site generated by UDG, creating single stranded regions which can be purified away using PCR purification spin columns or gel purification. See Figure 4.

A protocol for generating Bytes can be found here and more information on Byte formation can be found here.

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 Byte production (ie: USERTM) 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 USERTM-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. Once the desired number of Bytes 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 USERTM 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 USERTM treatment. I SceI site and A ends are highlighted