Gene synthesis is the technology of artificially synthesizing double-stranded DNA in vitro, and is a crucially important technique in molecular biology. Conventional gene synthesis generally involves assembling small segments of DNA and/or amplifying known samples of genetic material, and requires the raw nucleobases guanine, cytosine, adenine, thymine and uracil as starting materials.

GC-content (guanine-cytosine content) refers to the proportion of two nitrogenous bases, guanine and cytosine, in DNA and RNA molecules, which might be any domain of a gene, single gene, gene clusters, or even non-coding regions. Synthesis of high GC-content sequences can be troublesome due to issues with secondary structure, mispriming, or mis-annealing. GC-rich regions tend to facilitate base stacking, which makes them more stable than sequences with low GC-content. Additionally, secondary structures formed by high GC-content regions also tend to be stable and more resilient to denaturation. Sequences containing many guanine repeats can also generate complicated inter-strand folding due to hydrogen bonds between adjacent guanines.

GC-content is closely related to temperature optimization in gene synthesis. In PCR, primer GC ratio strongly influences the predicted annealing temperature of DNA templates. High GC-content tends to require a high melting temperature, which can result in mispriming or mis-annealing between the template and its complementary strand, leading to undesired and inaccurate gene products. Even when GC-rich sequences or G repeats are located in noncoding regions, the secondary structures formed can still affect factors in gene synthesis, such as melting temperature. Thus, an important part of gene synthesis is codon optimization, using synonymous codons to lower the GC-content of a sequence and lowering the melting temperature.

Synbio Technologies is proficient at accurately synthesizing error-free DNA constructs meeting customers’ requests, and is capable of assembling multi-kilobase plasmids or even entire genomes. Synbio Technologies can make gene synthesis perfect by handling even the most challenging synthesis requests, including genes or genomes with various complex sequences such as hairpin structures, high GC ratio, high AT percentage, multiple consecutive nucleotide sequences, and so on. The successful synthesis of GC-rich constructs is important to fully understand and study complex genes and even their non-coding segments with high conservation, which in turn poses profound significance in biomedical research.