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High-throughput synthesis of DNA fragments by molecular self-assembly of overlapping oligonucleotides
nature.com • • science
Key Points:
- A novel DNA assembly method using nicking endonuclease digestion and oligonucleotide overhangs enables efficient recombination of gene fragments with vectors, forming circular double-stranded DNA repaired in vivo in E. coli.
- The assembly method successfully constructs DNA fragments ranging from 300 bp to 3,000 bp, with a maximum fragment size of 9.9 kb demonstrated, though assembly efficiency decreases with larger sizes; using T4 ligase improves viability of clones up to 3 kb.
- The approach covers over 88% of human genes by targeting fragment sizes up to 3,000 bp, aligning with the typical length distribution of human coding sequences.
- Experimental validation includes one-pot and two-step assembly strategies for plasmids up to 9.9 kb, with high assembly efficiency confirmed by PCR, enzymatic digestion, and Sanger sequencing in both E. coli and yeast.
- Application of the method enabled creation of diverse combinatorial libraries, yielding novel fluorescent protein variants and PETase enzyme variants with enhanced fluorescence and enzymatic activities, supported by next-generation sequencing showing even library distribution and high complexity.
