Improved Methods for Rapid Gene Synthesis

摘要

Disclosed are methods and materials for assembling long polynucleotides from synthetic oligonucleotides. The use of synthetic oligonucleotides permits non-natural design of sequences. The oligonucleotides used for construction may be relatively short, according to practicalities of nucleotide synthesis. They are assembled using a ligase which is operative over a range of temperatures, i.e., is thermostable. The method and oligonucleotides are designed such that the melting temperature of the strands to be hybridized is set at a number of selected specific temperatures for each group of oligonucleotides to be hybridized and ligated. Hybridization and ligation take place at or near the melting temperature, so that each succeeding ligation is governed by a temperature that will prevent hybridization if any mismatches are present.