What is GC Content and Why Does It Matter?

GC content is one of the most basic yet important properties of a DNA sequence. It tells you the proportion of bases that are either guanine (G) or cytosine (C). This single number has a significant impact on how DNA behaves in the lab and in the cell.

How to Calculate GC Content

The formula is straightforward:

For example, if a 100 bp sequence contains 30 G bases and 28 C bases, the GC content is 58%. The remaining 42% is AT content.

Why GC Content Affects Melting Temperature

G-C base pairs are held together by three hydrogen bonds, while A-T pairs have only two. This means GC-rich sequences require more energy to separate the two strands. As a result, sequences with higher GC content have a higher melting temperature (Tm).

This relationship is used in the basic Tm formula for short oligonucleotides:

A primer with 60% GC content will have a noticeably higher Tm than one with 40% GC content, even at the same length. This matters when setting your PCR annealing temperature.

GC Content and Secondary Structure

High GC content increases the likelihood of intramolecular secondary structures such as hairpins and G-quadruplexes. These structures can block PCR amplification, inhibit reverse transcription, and interfere with sequencing reads.

Sequences with GC content above 70% are considered GC-rich and often require special PCR additives such as DMSO, betaine, or GC-specific polymerases to amplify reliably.

Typical GC Content Values by Organism

These values reflect the evolutionary pressures on each genome. Organisms living in hot environments often have higher GC content to stabilize their DNA at elevated temperatures.

GC Content in Primer Design

For PCR primers, the recommended GC content is 40% to 60%. This range provides a good balance between binding stability and specificity. Primers outside this range are more likely to cause problems:

• Below 35%: weak binding, low Tm, risk of non-specific amplification
• Above 65%: risk of secondary structures, high Tm, potential for non-specific priming

A GC clamp of 1 to 3 G or C bases at the 3' end of a primer improves binding at the extension site without pushing the overall GC content too high.

Sliding Window GC Analysis

Analyzing GC content across a sequence using a sliding window reveals local variation that a single average value misses. A window of 100 to 200 bp is common for identifying GC-rich and AT-rich regions.

This type of analysis is useful for:

• Identifying CpG islands near gene promoters
• Locating regions that may be difficult to amplify by PCR
• Predicting areas of high secondary structure potential
• Comparing genomic regions between species

GC Content and Codon Usage

In coding sequences, GC content at the third codon position (GC3) varies widely between organisms. Bacteria like E. coli prefer codons ending in G or C, while organisms like Plasmodium strongly prefer A or T at the third position. This has direct implications for codon optimization when expressing a foreign gene in a new host.

Summary

GC content is a simple number that carries a lot of information. It predicts melting temperature, secondary structure risk, amplification difficulty, and codon usage bias. Checking GC content is one of the first things to do when working with any new sequence.