Gregory Linshiz

Recursive construction of perfect DNA molecules from imperfect oligonucleotides.

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error‐free DNA molecules and their libraries from error‐prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem‐solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error‐prone oligonucleotides are recursively combined in vitro, forming error‐prone DNA molecules; error‐free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error‐free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

De Novo DNA Synthesis Using Single-Molecule PCR

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

Processing DNA molecules as text.

Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg’s movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing—the creation of variations and combinations of existing DNA—using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction.

Computer-aided high-throughput cloning of bacteria in liquid medium.

Bacterial cloning was first introduced over a century ago and has since become one of the most useful procedures in biological research, perhaps paralleled in its ubiquity only by PCR and DNA sequencing. However, unlike PCR and sequencing, cloning has generally remained a manual, labor-intensive, low-throughput procedure. Here we address this issue by developing an automated, computer-aided bacterial cloning method using liquid medium that is based on the principles of (i) limiting dilution of bacteria, (ii) inference of colony forming units (CFUs) based on optical density (OD) readings, and (iii) verification of monoclonality using a mixture of differently colored fluorescently labeled bacteria for transformation. We demonstrate the high-throughput utility of this method by employing it as a cloning platform for a DNA synthesis process.

Recursive Construction of Perfect DNA Molecules and Libraries from Imperfect Oligonucleotides

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology, and synthetic biology. We developed an error-correcting recursive construction procedure that attempts to address this challenge. Making DNA molecules from synthetic oligonucleotides using the procedure described here surpasses existing methods for de novo DNA synthesis in speed, precision, and amenability to automation. It provides for the first time a unified DNA construction platform for combining synthetic and natural DNA fragments, for constructing designer DNA libraries, and for making the faultless long synthetic DNA building blocks needed for de novo genome construction.

Programmable In Vivo Selection of Arbitrary DNA Sequences

The extraordinary fidelity, sensory and regulatory capacity of natural intracellular machinery is generally confined to their endogenous environment. Nevertheless, synthetic bio-molecular components have been engineered to interface with the cellular transcription, splicing and translation machinery in vivo by embedding functional features such as promoters, introns and ribosome binding sites, respectively, into their design. Tapping and directing the power of intracellular molecular processing towards synthetic bio-molecular inputs is potentially a powerful approach, albeit limited by our ability to streamline the interface of synthetic components with the intracellular machinery in vivo. Here we show how a library of synthetic DNA devices, each bearing an input DNA sequence and a logical selection module, can be designed to direct its own probing and processing by interfacing with the bacterial DNA mismatch repair (MMR) system in vivo and selecting for the most abundant variant, regardless of its function. The device provides proof of concept for programmable, function-independent DNA selection in vivo and provides a unique example of a logical-functional interface of an engineered synthetic component with a complex endogenous cellular system. Further research into the design, construction and operation of synthetic devices in vivo may lead to other functional devices that interface with other complex cellular processes for both research and applied purposes.

De novo DNA synthesis using single-molecule PCR.

The throughput of DNA reading (i.e., sequencing) has dramatically increased recently owing to the incorporation of in vitro clonal amplification. The throughput of DNA writing (i.e., synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning needs to be integrated into DNA synthesis methods. Here, we show how a new single-molecule PCR (smPCR)-based procedure can be employed as a general substitute for in vivo cloning, thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our previously described recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligonucleotides, entirely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used, in principle, in conjunction with other DNA synthesis methods as well.

Recursive construction and error correction of DNA molecules and libraries from synthetic and natural DNA.

Making error-free, custom DNA assemblies from potentially faulty building blocks is a fundamental challenge in synthetic biology. Here, we show how recursion can be used to address this challenge using a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone synthetic oligonucleotides and naturally existing DNA. Specifically, we describe how divide and conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide target DNA sequences into overlapping, albeit error prone, oligonucleotides, and how recursive construction is applied in vitro to combine them to form error-prone DNA molecules. To correct DNA sequence errors, error-free fragments of these molecules are then identified, extracted, and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. The method allows combining synthetic and natural DNA fragments into error-free designer DNA libraries, thus providing a foundation for the design and construction of complex synthetic DNA assemblies.