more favorable stacking energy for GC pairs than for AT or AU pairs because of the relative positions of exocyclic groups. Additionally, there is a correlation between the order in which the bases stack and the thermal stability of the molecule as a whole. Determination GC-content is usually expressed as a percentage value, but sometimes as a ratio (called G+C ratio or GC-ratio). GC-content percentage is calculated as whereas the AT/GC ratio is calculated as . The GC-content percentages as well as GC-ratio can be measured by several means, but one of the simplest methods is to measure the melting temperature of the DNA double helix using spectrophotometry. The absorbance of DNA at a wavelength of 260 nm increases fairly sharply when the double-stranded DNA molecule separates into two single strands when sufficiently heated. The most commonly used protocol for determining GC-ratios uses flow cytometry for large numbers of samples. In an alternative manner, if the DNA or RNA molecule under investigation has been reliably sequenced, then GC-content can be accurately calculated by simple arithmetic or by using a variety of publicly available software tools, such as the free online GC calculator. Genomic content Within-genome variation The GC-ratio within a genome is found to be markedly variable. These variations in GC-ratio within the genomes of more complex organisms result in a mosaic-like formation with islet regions called isochores. This results in the variations in staining intensity in chromosomes. GC-rich isochores typically include many protein-coding genes within them, and thus determination of GC-ratios of these specific regions contributes to mapping gene-rich regions of the genome. Coding sequences Within a long region of genomic sequence, genes are often characterised by having a higher GC-content in contrast to the background GC-content for the entire genome. Evidence of GC ratio with that of length of the coding region of a gene has shown that the length of the coding sequence is directly proportional to higher G+C content. This has been pointed to the fact that the stop codon has a bias towards A and T nucleotides, and, thus, the shorter the sequence the higher the AT bias. Comparison of more than 1,000 orthologous genes in mammals showed marked within-genome variations of the third-codon position GC content, with a range from less than 30% to more than 80%. Among-genome variation GC content is found to be variable with different organisms, the process of which is