Since the de novo
synthesis of the suppressor transfer RNA gene was first reported three decades ago 
, our ability to engineer and assemble synthetic gene constructs has revolutionized the field of biomedicine 
. Yet, despite our many achievements from assembling multi-kilobase plasmids to whole genomes 
, de novo
synthesis of GC-rich fragments remains a major obstacle namely because of secondary structure formation. Sequences populated with G repeats produce complex inter and intrastrand folding due to increased hydrogen bonding with neighboring guanines at their N-7 ring positions 
. In PCR, this phenomenon is marked by the appearance of shorter bands following gel electrophoresis. These truncated versions of the target amplicon are primarily the consequence of arrest sites (hairpins) introduced into the template causing premature termination to polymerase extension 
. In addition, mispriming and mis-annealing between template and compliment strands due to high melting temperature (Tm
) overlaps may contribute to incorrectly amplified gene constructs 
. Because of these complications, GC-rich sequences are typically optimized by the researcher using web-based tools 
that disrupt G repeats by choosing synonymous codons with lower Tm
s. However, there may be instances where nucleotide conservation is essential 
particularly for non-coding regions where secondary structure functions to activate or repress transcriptional initiation 
. While techniques are available to manage these difficult regions during PCR amplification of plasmid and genomic DNA 
, to our knowledge no method for de novo
synthesis of GC-rich templates has been clearly defined. The closest application we found was GeneDesign 
, which has the option to circumvent base rearrangement by adjusting the overlap between complimentary strands. While this can aid in ‘normalizing’ the overall Tm
of less GC-rich sequences, synthesis of longer oligodeoxynucleotides (ODN)s is often required, and may necessitate costly purification.
As a cheap and effective approach to disrupting secondary structure formation and minimizing high Tm
ODN overlaps in de novo
synthesis, we explored the use of the more popular and often referenced chemical agents, Dimethyl Sulfoxide (DMSO) 
and betaine 
during both the assembly and PCR amplification steps in conventional gene synthesis. These isostabilizing agents facilitate strand separation of double helix DNA by altering its melting characteristics. For example, betaine, an amino acid analog with both positive and negative charges close to neutral pH, acts to equilibrate the differential Tm
between AT and GC base pairings; DMSO on the other hand, acts by disrupting inter and intrastrand re-annealing.
In this study, we compared the effects of these additives in the construction of two GC-rich gene fragments implicated in tumorigenesis, the Insulin-like Growth Factor 2 Receptor (IGF2R) 
and V-raf murine sarcoma viral oncogene homolog B1(BRAF) 
. DMSO and betaine were also chosen because of their previously reported success in PCR amplification of the IGF2R gene fragment from a vector 
. However, for our purposes, IGF2R and BRAF were chemically synthesized and assembled in vitro
by pooling overlapping, single-stranded ODNs using two conventional methods, the Polymerase Chain Assembly (PCA) 
and the Ligase Chain Reaction (LCR) 
. For a typical PCA reaction, assembly is done with one or two pre-PCR steps where single-stranded ODNs prime off each other, building up to the full-length product; 40 bp ODNs are designed (no gaps) with 20 bp overlap between template and compliment strands where a 3′ recess allows for polymerase binding and strand propagation. ODNs for LCR are the same as those for PCA except that each strand is 5′ phosphorylated for ligation. In this case, complimentary ODNs are denatured and annealed over several cycles for optimum strand alignment. A final round of PCR is then employed in both methods to amplify the target product using outside primers.
Here we report that DMSO and betaine greatly improve de novo synthesis of IGF2R and BRAF gene fragments generated from both PCA and LCR methods of assembly. Though we only tested two genes, incorporation of either additive could aid in the construction of most GC-rich sequences. Protocol manipulation of standard conditions is also unnecessary due to the isostabilizing properties of these additives. Even without the need for nucleotide conservation, this application saves a great deal of end-user time not having to re-design and codon optimize ODNs prior to synthesis. As such, the possibility of manually introducing sequence error is also limited; one mismatch, deletion or insertion could lead to a frame-shift or other gene lethality. Furthermore, DMSO and betaine are very inexpensive, easily obtainable and highly compatible with other biological agents, which make them ideal for any gene synthesis assay.