DNA synthesis, assembly and applications in synthetic biology

Abstract

The past couple of years saw exciting new developments in microchip-based gene synthesis technologies. Such technologies hold the potential for significantly increasing the throughput and decreasing the cost of gene synthesis. Together with more efficient enzymatic error correction and genome assembly methods, these new technologies are pushing the field of synthetic biology to a higher level.

Figure 1

Gene assembly strategies from microarray derived oligonucleotides. a) Oligonucleotides synthesized on a microarray are less expensive, but the high heterogeneity and error rate requires appropriate retrieval and segregation technologies to assembly them into gene constructs. b) Next generation sequencing was used to identify error-free oligo sequences for gene assembly. c) Selective amplification of oligonucleotides from the pool. d) Physically dividing a microarray into isolated sub-arrays. Oligonucleotides are amplified and assembled into gene fragments on chip within each reaction well.

Figure 2

Schematic illustration of oligonucleotide error-correction by next generation sequencing. Microarray synthesized oligonucleotides are eluted and amplified before they were fed into a 454/Roche pyrosequencing instrument. The desired sequences are identified, sorted and retrieved with a microactuator-controlled micropipette, and PCR-amplified into full-length gene constructs [14].

Figure 3

Schematic illustration of gene assembly by selective amplification of microarray oligonucleotides. Oligonucleotides are eluted from the microchips to yield a heterogeneous pool. Specific short PCR primers are designed to selectively amplify oligonucleotides from the pool (hot and pale pink). Further selective amplification with end primers (shades of blue) generates assembly pools containing sequences for a single gene, which are then assembled in to gene constructs by PCR reactions [16].

Figure 4

Schematic illustration of on-chip DNA amplification and gene assembly. The microchips are divided into physically isolated sub-arrays where oligonucleotides are amplified by on-chip strand displacement amplification. The released strands are the assembled in to 0.5–1 kb gene fragments within the wells [40].