Much work has been done to try and increase the efficiency by which we generate the Bytes, anchor them, assemble them, and terminate them. A general outline of the optimizations we have considered and worked on are shown below as well as their effects on the process.

The original format of the universal primers did not have the uracils distributed evenly within the primer. The result was poor efficiency in construction on a bead. Our hypothesis was that the uracils, if they were distributed more evenly, would create smaller pieces of ssDNA that would more easily melt off the Byte to generate fully ssDNA 12 base overhangs. The first version of our USER ends is shown below. By changing primers to their current form we have consequently increased efficiency of construction 2.5 times that of the first version.

The first step in producing workable quantities of BioBytes is PCR with the universal deoxyuracil-containing primers. Only slight tweaking of PCR conditions was required to produce ideal quantities of Bytes.

• The Primers

  • HPLC purified primers were ordered and PCR was attempted with them. The motivation for this was the possibility that during oligo synthesis only a fraction of the synthesized primers are full length. We thought that this may have an effect on the Byte end generation. Unfortunately not only were the primers very expensive to have HPLC purified and that they were in very low yield, but also that the resulting Bytes showed no improved efficiency in ligation.

• The dNTPs

  • Concentration effects of dNTPs were considered. We ran six PCR reactions in total: 3 sets for each AB/BA primer pair. The three conditions tested were 0.2, 0.6, 0.8, 1.2, and 1.5 µM final concentration of dNTPs. Optimal PCR yield was at 0.2 µM dNTPs.

An essential step in assembly with BioBytes if the preparation of the Bytes. Following PCR the product is USER^TM digested to nick the DNA. Finally, the Bytes are purified away from these small ssDNA pieces to prevent their binding to the sticky ends during assembly and consequently negatively influencing the efficiency of construction. The following describes the results of optimization experiments conducted to increase efficiency of BioBytes.

• USER^TM Digestion

  • The amount of USER^TM in the digest was investigated by digesting two sets of BA/AB Bytes: either 4% or 10% total volume of USER^TM was added to the PCR product tubes. The 4%, for instance, corresponds to 1 µL of USER^TM mix in a 25 µL PCR reaction. The conclusion was that slight improvement in ligation efficiency was seen for 10% over 4% USER^TM addition.
  • USER^TM digestion duration was considered at 0.5, 1, 1.5, and 2 hours. By examining the efficiency of ligation of AB and BA Bytes processed under these conditions that no changes in efficiency were observed after 1 hour.
    
  • PCR purification conditions were considered in terms of the temperature at which the purification, using a spin column kit, was conducted. 23°C(room temperature), 37°C and 50°C conditions were tried. We observed maximal efficiency in terms of yield and ligation efficiency of Bytes by PCR purifying at 37°C.
    
  • Alternative purification techniques proved better than PCR purification. We found that by running the USER^TM digested PCR products on a gel then gel extracting the band of interest not only prevented carry over of template DNA from PCR and extraneous PCR products (minimal anyway), but also showed better efficiency for ligation of the Bytes. To optimize even further we found that adding pH 5.2 3M sodium acetate to the dissolved gel pieces gave increased yields.
    
  • Ligation is an important part of Byte assembly on a bead. We tried both T4 and Taq DNA ligases and found that by far T4 was better. T4's increased efficiency allowed large constructs to be assembled with higher relative yields. We also tried ligation at room temperature versus at 4°C, hypothesizing that at reduced temperature ligation efficiency will go down but the 3' 12 base Byte ends may anneal more stably. We ligated for nearly 1.5 hours under both conditions and found that ligation at room temperature is preferable.