Successful preparation and analysis of a 5-site 2-variable DNA library

DNA code sequences generated by the program SynDCode were used to construct a 5-site, 2-variable computational DNA library by parallel overlap self-assembly. The final library was amplified using the polymerase chain reaction (PCR) to obtain a fragment of the expected size. Twelve library sequences randomly selected from the library by cloning were sequenced and found to be distinct and correctly assembled library strands. A Birthday Problem-like analysis suggests that we have all 32 different molecules in our library mixture. We then developed new protocols using DNA hybridization to successfully identify single members of this library. We have also used this protocol to analyze mixtures of clones from the library. This approach shows the experimental validation of the ability to distinguish different sequences generated from the SynDCode program. We are in the process of working out protocols to separate out specific library members and to expand this library.