RNA-Mediated Gene Assembly from DNA Arrays

摘要

The widespread availability of peptides and oligonucleotides synthesized by solid-phase methods has had a profound impact upon biology and medicine, with a myriad of important uses in research, diagnostics, and therapeutics. A limitation of current technologies is the relatively short length of the molecules that can be synthesized, as determined by the stepwise reaction yield, and thus peptides and oligonucleotides are usually restricted to lengths below approximately 50 amino acids or 100 nucleotides (nt), respectively. This synthetic limitation has driven interest in the development of alternative approaches for the production of full-length genes and proteins. The most common strategy has been to splice together shorter segments into a full-length, functional assembly; for example, the Staudinger ligation reaction permits full-length proteins to be constructed from a series of peptides, and full-length genes can be obtained from multiple short single strands in a series of sequential ligation steps or by polymerase cycling assembly (PCA). However, the assembly based strategies for gene synthesis reported to date remain laborious, expensive, and time-consuming, and thus have not yet provided the level of accessibility needed for widespread utility.