Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

Abstract

The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read-write cost gap.

Figure 1.

The synthetic biology test cycle. (From top, clockwise) Synthetic DNA constructs are designed and manipulated using computer-aided design software. The designed DNA is then divided into synthesizable pieces (synthons) up to 1–1.5 kbp. The synthons are then broken up into overlapping single-stranded oligonucleotide sequences and chemically synthesized. The oligonucleotides are then assembled together into the designed synthons using gene synthesis techniques. If necessary, multiple synthons can be assembled together into larger DNA assemblies or devices. The assembled DNAs are then typically cloned into an expression vector and sequence-verified. Once verified, the synthetic constructs are transformed into a cell and the function of the synthetic construct is assayed. Depending on the results the constructs can then be modified or refined and the test cycle is repeated until a DNA construct is obtained that produces the desired function.

Figure 2.

Phosphoramidite-based synthesis of oligonucleotides. This synthesis process is the most commonly used for the synthesis of DNA oligonucleotides for gene synthesis.

Figure 3.

Methods for solid-phase synthesis of oligonucleotides. (A) Column-based oligonucleotide synthesis. This is the traditional method of synthesizing DNA using solid-phase phosphoramidite chemistry. The synthesis of a unique oligonucleotide sequence is done on the surface of controlled-porosity glass beads (CPG) contained with a synthesis column. During the synthesis process, the reagents flow through the column and across the packed CPG matrix and the oligonucleotide “grows” off from the bead surface. Only one sequence can be synthesized per column, although high-throughput synthesizers exist that can synthesize on multiple columns at once. A 96-column synthesis plate is shown as an example. (B) Microarray-based oligonucleotide synthesis. In this method, microarray chips containing tens of thousands of distinct features synthesize unique oligonucleotide sequences at once with one unique oligonucleotide sequence synthesized per chip feature. On standard arrays, there are no physical barriers between features, so following cleavage of the synthesized oligonucleotides from the chip surface the end product is a pool of sequences containing every oligonucleotide synthesized on the array. Subsequent processing steps are required to “fish” the desired oligonucleotide sequences out of the synthesis pool for subsequent gene synthesis. The OligoArray CMOS microarray chip is shown as an example, although a handful of different array formats exist for oligonucleotide synthesis.

Figure 4.

Polymerase chain assembly. Overlapping oligonucleotides encoding a DNA duplex are assembled together via progressive overlap extension assembly in a one-pot reaction. Following assembly the full-length assembled synthon is amplified out of the assembly mixture by polymerase chain reaction (PCR) with the outermost primers.

Figure 5.

Gene synthesis from microarray-synthesized oligonucleotides. (A) Synthons can be assembled using on-chip synthesis and assembly by including a single priming site into the 3′-end of every oligonucleotide synthesized on the microarray. The oligonucleotides can then be amplified within microwells designed into the array by incubating with a common primer and a DNA polymerase. The primer sequence is removed from the assembly oligonucleotides using a nicking endonuclease, freeing the oligonucleotides to be assembled together via polymerase chain assembly within the same well. (B) Synthons can also be synthesized off-chip by first cleaving the oligonucleotide pools from the array. The cleaved synthesis oligonucleotide pool can be amplified into subpools by PCR using priming sites incorporated into both ends of the synthesized oligonucleotide. The amplified subpools can then be further subdivided into assembly oligonucleotide pools by additional unique priming sites included in the oligonucleotide flanking sequences. Following segregation by amplification the priming sequences are removed from the assembly oligonucleotides by restriction enzyme digestion. The processed oligonucleotides can then be assembled together by polymerase cycle assembly.