How to Use
Vector Design Studio
GC Content Calculator
GC content of DNA templates is a critical factor which determines the success of cloning target genes into desired backbones. Gene templates with high GC content often result in higher chances of forming self-dimers or secondary structures and require higher annealing temperatures. VectorBuilder can help with cloning sequences ranging from simple to complex.
This tool allows you to determine the GC content of entire gene sequences as well as specific regions within a gene. When a DNA or RNA sequence is entered, it calculates both the number and percentage of each base type, including degenerate bases. In addition, our tool features a graphical readout, enabling direct visualization of GC content and prediction of CpG islands.
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).
GC content in PCR
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. 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%.
CpG island
A CpG island (CGI) is a GC-rich DNA region where the CpG dinucleotide frequency is significantly higher than the surrounding genome. It is defined as being at least 200-base-pairs long, with a GC content of 50% or higher and a CpG observed-to-expected ratio greater than 0.6. CGIs are almost completely unmethylated and are often associated with promoter regions regulating transcription initiation in vertebrates. Predicting CGIs within a given sequence can flag potential transcription start sites, highlight regulatory hot spots, or guide DNA methylation studies. In addition, knowing CGI boundaries helps improve your experimental designs in molecular cloning, CRISPR editing, or other applications that are impeded by high GC content or DNA secondary structures.
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 directly to your cloning experiment in the Vector Design Studio.