Team:Alberta/DNAanchor

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<p>The current method for Byte production (ie: USER<sup>TM</sup>) necessitated a particular anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER<sup>TM</sup>-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 USER<sup>TM</sup> 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 <i>E. coli</i> genome. See <B>Figure 1</B>.</P>
<p>The current method for Byte production (ie: USER<sup>TM</sup>) necessitated a particular anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER<sup>TM</sup>-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 USER<sup>TM</sup> 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 <i>E. coli</i> genome. See <B>Figure 1</B>.</P>
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<p>A vital component of the BioBytes method is the use of a biotinylated DNA anchor piece in order to allow unidirectional assembly of the Bytes on a bead.  The anchor itself has three vital components:  A 5’ biotinylation, a dsDNA portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the anchor.  Our team has considered a number anchor systems, each with their own set of advantages and disadvantages.  The three main anchor systems we investigated are summarized below.</P>
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<p>A vital component of the BioBytes method is the use of a biotinylated DNA anchor piece in order to allow unidirectional assembly of the Bytes on paramagnetic beads.  The anchor itself has three vital components:  A 5’ biotinylation, a dsDNA portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the anchor.  Our team has considered a number anchor systems, each with their own set of advantages and disadvantages.  The three main anchor systems we investigated are summarized below. See <B>Table 1</B>.</P>
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<p><B>Figure 1:</B> pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions.<p>
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<p><B>Table 1:</B> Overview of three different anchoring systems that were considered for BioBytes.<p>
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Revision as of 06:07, 21 October 2009

University of Alberta - BioBytes










































































































DNA Anchor/Terminator

Anchoring System

The current method for Byte production (ie: USERTM) necessitated a particular 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 1.

A vital component of the BioBytes method is the use of a biotinylated DNA anchor piece in order to allow unidirectional assembly of the Bytes on paramagnetic beads. The anchor itself has three vital components: A 5’ biotinylation, a dsDNA portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the anchor. Our team has considered a number anchor systems, each with their own set of advantages and disadvantages. The three main anchor systems we investigated are summarized below. See Table 1.


Table 1: Overview of three different anchoring systems that were considered for BioBytes.



Figure 1: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions.