Programmable iterated elongation: a method for manufacturing synthetic genes and combinatorial DNA and protein libraries

A method for at least semi-automatically manufacturing a biopolymer, comprising:

• a. analyzing the biopolymer to be constructed to determine a plurality of potentially faulty subcomponents for collectively defining the biopolymer;
• wherein said subcomponents are selected from the group consisting of nucleotides, polynucleotide, oligonucleotide, double stranded or single stranded DNA or RNA, a synthetic gene, an oligonucleotide of at least about 100 bases in length, an oligonucleotide of at least about 200 bases in length, an oligonucleotide of at least about 400 bases in length, or combinations thereof;
• b. determining a hierarchical process for recursively constructing the biopolymer from said potentially faulty subcomponents, said determining comprising
• decomposing said hierarchical process into a plurality of sub-processes by searching for an optimal and valid division point of said hierarchical process, and then selecting an optimal set of sub-processes to construct the biopolymer for said hierarchical process, said hierarchical process comprising a plurality of stages, each stage comprising a plurality of sub-processes such that an output of said sub-processes of said stage is a plurality of sub-components to be combined as an output of said stage, wherein said output of a previous stage is an input to a subsequent stage of said hierarchical process, wherein a first stage and a first plurality of sub-processes are defined according to said division point, which determines a starting point for said hierarchical process, such that all subsequent stages and sub-processes are determined according to said first stage and said first plurality of sub-processes;
• wherein said sub-processes are collectively optimized and wherein at least one sub-process for combining a plurality of subcomponents is selected according to one or more criteria selected from the group consisting of:
• time, energy input, by-products, monetary expense, number of subcomponents, sub-processes required, and any combination thereof, wherein said valid division point is at least partially determined according to at least one constraint selected from the group consisting of availability of at least one subcomponent, characterization of at least one subcomponent and cost of performing an associated sub-process;
• wherein said hierarchical process is determined at least partially according to an optimal protocol wherein said optimal protocol is determined based on a set of constraints and a set of cost parameters wherein said set of constraints comprises a plurality of subcomponents to be synthesizable as oligo-primers, size of oligonucleotides, number of oligonucleotides, number of reactions, number of sub-processes, number of levels in the construction hierarchy, availability of oligonucleotides having defined sequences, and use of natural DNA or RNA;
• and
• c. constructing a set of instructions for assembling the biopolymer according to said plurality of potentially faulty subcomponents and according to said hierarchical process;
• and
• d. recursively constructing the biopolymer according to said set of instructions from a plurality of subcomponents, wherein at least one subcomponent has an error and wherein at least one sub-process of said hierarchical process corrects said error providing a final biopolymer product not featuring an error, wherein said biopolymer is a polynucleotide or oligonucleotide;
• wherein said subcomponents comprise single stranded DNA and at least one oligonucleotide primer, and said plurality of sub-processes comprise elongation, phosphorylation and degradation by a DNA degrading enzyme, such that said recursively constructing the biopolymer comprises performing an elongation reaction with two input overlapping ssDNA fragments to form a dsDNA;
• phosphorylating a primer at the 5′ end;
• amplifying the dsDNA with said primer;
• and degrading said phosphate-labeled PCR strand with said DNA degrading enzyme, yielding an elongated ssDNA fragment as output;
• wherein said stages are repeated to form said biopolymer.