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Abstract

This invention provides processes to assemble many (greater than 20) partially overlapping single stranded DNA molecules (fragments) having preselected sequences, followed by extension of those strands that hybridize at terminal overlap regions, and ligation of the extend products, creating a double-stranded DNA assembly. These processes use non-standard nucleotides carrying heterocyclic nucleobase analogs that implement non-standard hydrogen bonding patterns; these allow controlled annealing of the single stranded fragments via Watson-Crick rules, with less or no interference from a range of non-Watson Crick interactions, hairpin formations, or off-target hybridization displayed by standard nucleobases. This process includes an optional conversion step that replaces non-standard nucleobase pairs with standard nucleobase pairs, generating large synthetic DNA (LS-DNA) molecules containing only natural nucleotides. As useful application, this invention allows the assembly of genes encoding whole proteins (typically 1000-3000 nucleotide pairs) from a collection of single stranded DNA fragments at reduced cost and effort.

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First Claim