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

Abstract

A method for manufacturing synthetic genes and combinatorial DNA and protein libraries, termed here Divide and Conquer-DNA synthesis (D&C-DNA synthesis) method. The method can be used in a systematic and automated way to synthesize any long DNA molecule and, more generally, any combinatorial molecular library having the mathematical property of being a regular set of strings. The D&C-DNA synthesis method is an algorithm design paradigm that works by recursively breaking down a problem into two or more sub-problems of the same type. The division of long DNA sequences is done in silico. The assembly of the sequence is done in vitro. The D&C-DNA synthesis method protocol consists of a tree, in which each node represents an intermediate sequence. The internal nodes are created in elongation reactions from their daughter nodes, and the leaves are synthesized directly. After each elongation only one DNA strand passes to the next level in the tree until receiving the final product. Optionally and preferably, error correction is performed to correct any errors which may have occurred during the synthetic process.

1 . A system for at least semi-automatically manufacturing a polymer, comprising:
 (a) a first computational device for operating a language program for specifying a plurality of polymers and for deconstructing said plurality of polymers into a plurality of potentially faulty subcomponents of said plurality of polymers according to a hierarchical deconstruction protocol, such that the polymers are constructable from said plurality of potentially faulty subcomponents according to a hierarchical construction protocol;   (b) a second computational device for operating a program for constructing said polymers according to said hierarchical construction protocol, said program directing synthesis of a plurality of polymer molecules from said potentially faulty subcomponents and said program forming a robot control program for executing synthesis of the polymers, wherein said robot control program comprises said hierarchical construction protocol;   (c) a database for storing a plurality of synthetic schemes for synthesizing said polymers, a list of subcomponents for synthesizing said polymers, a model of the biochemical process of said polymer synthesis for determining cost of synthesis of the polymers, wherein said program determines said cost according to said model, selects one of said synthetic schemes according to said cost and directs the synthesis of the polymers;   (d) a storage for storing a library of potentially faulty subcomponents, comprising physical molecules corresponding to said plurality of potentially faulty subcomponents from said deconstructing by said first computational device; and   (e) a robot for being controlled by said robot control program of said second computational device and for synthesizing the polymer from said polymer molecules and said library of potentially faulty subcomponents according to said hierarchical construction protocol, wherein said hierarchical construction protocol potentially comprises an error correction protocol.   
     
     
         2 . (canceled) 
     
     
         3 . The system of  claim 1 , wherein said graphical language program performs:
 determining at least one mathematical rule for describing the library;   determining a plurality of subcomponents for collectively defining the library of polymers; and   constructing a set of instructions for assembling the library of polymers according to said plurality of subcomponents;   wherein said robot performs said set of instructions for synthesizing the library of polymers, wherein said synthesizing is performed at least semi-automatically.   
     
     
         4 . (canceled) 
     
     
         5 . The system of  claim 1 , wherein said hierarchical construction protocol further comprises:
 at least semi-automatically combining said at least two subcomponents according to at least one subprocess.   
     
     
         6 . (canceled) 
     
     
         7 . The system of  claim 1 , wherein said hierarchical construction protocol further comprises recursive construction according to said hierarchy, wherein said recursively constructing comprises modified recursion. 
     
     
         8 - 11 . (canceled) 
     
     
         12 . The system of  claim 7 , wherein said program further performs:
 recursively analyzing synthesis of the polymer to form a plurality of sub-sets of subcomponents for each set of subcomponents; and   independently determining synthesis of each sub-set of subcomponents.   
     
     
         13 - 16 . (canceled) 
     
     
         17 . The system of  claim 3 , wherein said graphical language program further performs:
 analyzing the plurality of final objects to detect at least one shared subcomponent between the final objects; and   determining a recursive, hierarchical construction process with a minimal number of subprocesses by using said at least one shared subcomponent.   
     
     
         18 . The system of  claim 3 , wherein said graphical language program further performs:
 analyzing the plurality of final objects to detect at least one shared subcomponent between the final objects;   analyzing the plurality of subcomponents to detect each error free part of each subcomponent; and   determining a recursive, hierarchical construction process with a minimal number of subprocesses by using said at least one shared subcomponent according to each error free part.   
     
     
         19 . The system of  claim 18 , further comprising:
 constructing a plurality of error free subcomponents from said subcomponents; and   constructing the plurality of final objects from said plurality of error free subcomponents.   
     
     
         20 . The system of  claim 1 , 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. 
     
     
         21 . The system of  claim 20 , further comprising a sequencing device, a PCR device and a purification device for directing the performance of said robot, wherein the synthetic process comprises a plurality of subprocesses, each subprocess includes PCR amplification with PCR device, followed by sequencing with sequencing device to look for one or more errors; wherein said purification device operates between the performance of subprocesses or after the one or more sequences have been synthesized. 
     
     
         22 . The system of  claim 21 , further comprising a measurement device in communication with said robot to monitor an amount of biopolymer being synthesized. 
     
     
         23 . The system of  claim 21 , wherein said graphical language program performs the following:
 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.   
     
     
         24 . The system of  claim 23 , 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.