Nucleotide bonds

In DNA and RNA, the distribution of bases has a significant impact on our ability to study and manipulate genes. Within the double helix, adenine always binds to thymine (or uracil in RNA) using two hydrogen bonds, while guanine binds to cytosine using three hydrogen bonds (Figure 1). The higher the number of hydrogen bonds, the more energy required to separate the two nucleotide bases. Therefore, GC pairs will have higher stability than AT pairs. The percentage of GC base pairs in genomes is highly variable, but most species range from 30-60%, with humans averaging 41%.

Figure 1. Binding of nucleotides across DNA strands (A), with highlighted hydrogen bonds for thymine-adenine pairs (B) and cytosine-guanine pairs (C).

Applications of GC content

When considering a fragment of DNA, for primer design or cloning of an entire gene, it is important to note GC content given the higher amount of energy required to break the three hydrogen bonds. Polymerase chain reaction (PCR) is a widely utilized tool where GC content is of high importance. DNA strands must be broken apart (denatured), and primers must be able to bind to the correct position on the DNA strand (annealing) for amplification. The distribution of nucleotides can influence both processes: DNA templates with high GC content may require higher denaturation temperatures, and high GC content in primers necessitates higher annealing temperatures. The optimal GC content for primers is around 50-55%, but high amplification can be achieved with GC content between 40 and 60%.

Determining the GC content of an entire sequence as well as distribution within a sequence is a valuable tool when designing and troubleshooting experiments. In addition to performing GC content analysis here, a GC content calculator is also built into our Codon Optimization and shRNA Design tools. Both applications can be explored on our Tools page and can be applied to directly to your cloning experiment in the Vector Design Studio.