Error-free building blocks for rapid and efficient assembly of biobricks

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Yusuf Murgha, Ivy Tran, Becky Ward, Erdogan Gulari, Jean-Marie Rouillard

MYcroarray, United States

DNA oligonucleotides serve as highly customizable building blocks for the assembly of biobricks, large synthetic DNA fragments or whole genomes. However oligonucleotide synthesis technologies, whether producing one sequence at a time or large pools of multiple sequences, are prone to errors. Therefore current assembly methods require cumbersome sequence verification steps to correct for frequent synthesis errors. Here we present the advantage of using error-free oligonucleotides as starting blocks for de novo assembly of large DNA fragments. We have developed a method to produce collections of individually separated, error-free long oligonucleotides from massively parallel synthesis on chips. We demonstrated by deep sequencing that post purification, nearly 100% of the molecules recovered for each individual sequences are perfect. We also show that these error-free building blocks can be efficiently assembled into larger fragments and mitochondrial genomes. And with a size of up to several hundred bases, they can be considered as custom synthetic biological parts ready for integration into complex molecular circuitry. The availability of these inexpensive error-free building blocks will undoubtedly accelerate the pace of research to advance the field of synthetic biology.