It is not yet possible to synthesize entire genes as long continuous strands of DNA from scratch. Rather, all synthetic genes are assembled from short custom made single stranded DNA oligonucleotides or “oligos”, which are literally strings of a few nucleotides. Oligos are by-and-large still synthesized the same way as they were 15 or 20 years ago. Through incremental improvements in instrumentation and higher throughput, oligos have become a cheap commodity for use in standard recombinant DNA technologies. But, more than anything else, great demand and even greater competition by manufacturers has driven the oligo prices down by about 10-fold over the past 15 years (). In comparison, the prices of finished, sequence confirmed, gene synthesis by commercial gene foundries have plummeted 50 fold in only 10 years (). As a reference point, at the outset of the poliovirus synthesis project  in 1999 commercial gene synthesis was simply unheard of. As recently as 2000, after much searching, we found a vendor who agreed to synthesize parts of the genome by special arrangement at a price of $12/bp (Cello, Paul, and Wimmer, 2002
Figure 2 Price development of oligonucleotide synthesis and de novo gene synthesis. Shown are the approximate end user prices per base for oligonucleotides (desalted, non-purified) or per base pair for synthetic genes (below 3kb, sequence guaranteed). The data (more ...)
In the ideal world, an efficient and economical de novo gene synthesis platform would combine cheap error-free oligo synthesis with accurate assembly methods. Neither one are currently available. There are two dramatically different methods of synthesiszing oligos. In the traditional, time-proven, method of solid-phase oligo synthesis each oligo is synthesized individually, on a separate small column or a well on a multiwell plate. The method is high yielding but costly ($ 0.10–0.20 per nucleotide synthesis cost), which is a critical aspect if the oligos are needed for the assembly of long DNA sequences. The price given above translates into an oligonucleotide cost of approximately $ 200–400 for a 1kb DNA sequence and that’s for the raw material only.
The development of optical deprotection chemistries heralded a new era of parallel synthesis methods on micro biochips (Fodor et al., 1991
) that can be used for both oligo or peptide synthesis. Depending on the chip platform being used, several thousands to hundreds of thousands of distinct oligonucleotides can theoretically be synthesized on a single chip.
In an ingenious extension Tian and collegues (Tian et al., 2004
) mated the light-induced deprotection chemistry with microfluidic technology that allows the programmable synthesis of thousands individual oligonucleotides on a tiny chip (). At the heart of this method is the Digital Light Processing technology (DLP) that was developed for digital projectors and High Definition Projection TV sets. On a microfluidic chip containing a labyrinth of thousands of connected tiny reaction chambers (), each chamber is computer-addressable by a light beam generated on a digital micromirror device (Singh-Gasson et al., 1999
) (akin to the individual color light spots making up the projection-TV picture). A DNA synthesis mixture containing the first nucleotide (A, for instance) is pumped through the system. Here, A only “sticks” to the chambers which call for an A at the specific position in their sequence, which are the ones that are being illuminated at that time (). Although all chambers receive the same synthesis mixture at any given times, no reaction occurs in the chambers that are “left in the dark” (in the example above, the ones that need a C, G, or T at their corresponding position). After the first reaction, the A-mix is washed out and the next reaction mix, containing the next nucleotide is pumped in and the process is repeated, four times in total. After all four nucleotide reaction mixes have gone through the chip, in each chamber the oligonucleotide chain has now grown by at least one nucleotide of the desired sequence.
Figure 3 Microfluidic chip technology coupled with light activated chemistries hold great promise for the massive parallel synthesis of oligonucleotides. (A) On an array of tiny flippable mirrors, each mirror can be separately computer-controlled (flipped to an (more ...)
At the end of the reaction the oligonucleotides are eluted from the chambers as a single pool. Each of the oligo sequences is only present in minute quantities. This may present a challenge in further increasing the throughput by increasing the number of reaction chambers per chip, while decreasing chip size. Tian et al. demonstrated the potential power of this technology for the synthesis of large numbers of oligonucleotides to be used in synthetic gene assembly (Tian et al., 2004
Companies already offer parallel on-chip-synthesized custom oligo mixtures that are amenable for gene synthesis (LC Sciences, Houston Texas). Currently the price of a pool of 3,912 90-mers is approximately $1000. This technology is still very much in the exploratory stage. One inherent difficulty of the method is that all oligos are released from the chip as a mixture. The low yields of oligos that come off the chip (107
molecules per sequence) are insufficient to drive a gene assembly reaction, which mandates a post-synthesis PCR amplification step before oligos can be used. For this purpose each oligo is synthesized with two flanking generic adaptor sequences, which allows amplification of all oligos in parallel in a single PCR reaction using the corresponding adaptor primer pair () (Tian et al., 2004
). Using distinct sets of adaptors on distinct subsets of oligos in the same chip-synthesis reaction allows the subsequent selective amplification of a desired subset of oligos, for instance a set necessary for the assembly of one particular gene. Therefore, it is possible that in a separate reaction a different set of oligos can be amplified from the same chip-eluted oligo mix. Thus fractioning the entire oligo pool into gene-specific subsets will reduce complexity of the mixture, increase concentration of each specific oligo, and reduce potential interference or cross-hybridization from other oligos in the pool. This will be especially useful as the number of individual sequences synthesized on the chip increases. The higher the number of discrete oligo sequences synthesized per chip, the lower the absolute yield per oligonucleotide (sub fmole range) because the total yield of DNA is a direct function of the total reaction surface on the chip. With more distinct oligos the potential for unwanted cross-hybridizations during the gene assembly step also increases.
Figure 4 Assembly of gene sequences from chip-synthesized oligonucleotides. The pool of overlapping oligos in minute amounts is released from the microchip, followed by PCR amplification with universal adapter primers. Double strand copies produced in this way (more ...)
The second drawback of the chip-based oligo synthesis is that the PCR amplified oligos are now in a double stranded form. The presence of a perfectly matched antisense strand may reduce the efficiency in the subsequent assembly of these oligos into larger genes. The assembly reaction depends on the complementarity of the overlapping “construction” oligos, those designed to build the gene, and the antisense oligos are likely to compete more effectively for the same hybridization partner. To overcome this problem the desired single stranded construction oligos can be selectively enriched by specific hybridization to antisense selection-oligos affixed to a column and subsequent elution (Tian et al., 2004
). When done under stringent enough conditions this procedure also contributes to a significant elimination of error-containing oligos, as they produce mismatches with the selection oligo and consequently elute from the column at a lower temperature. On the downside, this method requires twice the amount of selection oligos than there are contruction oligos. In other words, to produce one chip’s worth of oligos one needs two additional chips’s worth of selection oligos, tripling the cost of synthesis (Tian et al., 2004
). This brings the current “rock-bottom” cost of the final construction oligos before the gene assembly to about $0.03/bp.
While these new multiplex synthesis systems are technically feasible it is our understanding that the major suppliers of large synthetic DNA for now continue to assemble genes from individually synthesized overlapping oligonucleotides by traditional methods.
The sheer number of different oligonucleotides synthesized on a chip mandates the use of new software programs to handle the complexity of possible interactions of the various oligo sequences in the mix (Czar et al., 2009
). Several software programs are freely available to design optimal sets of assembly oligonucleotides. The basic tasks that successful software needs to perform are:
- Breaking down the target sequences to be synthesized into suitable overlapping oligos.
- Designing hybridization units, the overlapping portion between two oligos, with the same melting temperature.
- Ensuring hybridization specificity of each oligo pair to eliminate potential cross-hybridization by choosing the best possible breaking points between oligos for a particular gene, and by altering synonymous codons.