Quantification of size distribution of restriction fragments in mitochondrial genome using fluorescence correlation spectroscopy

A crucial investigation is to quantify restriction fragment length polymorphisms without gel electrophoresis, as the distribution of fragment size is mainly evaluated on the gel, which cannot be easily quantified. We developed a method to determine the fragmentation of the mitochondrial genome caused by restriction enzymes using fluorescence correlation spectroscopy (FCS). Distribution of fragment size was evaluated by the decrease in amplitude of the fluorescence correlation function while the mitochondrial genome PCR product was digested with Hga I or Hae III. Using a multicomponent model, which was considered as a fragment length-weighted correlation function, we calculated the correlation amplitude theoretically expected and compared it to that measured by FCS. These amplitudes for Hga I were coincident, whereas the measured amplitude for Hae III was more than the theoretical one. Because of tetra-nucleotide recognition by Hae III, there were many more fragments than with Hga I. Therefore, the amplitude measured by FCS would be a very useful index for primary screening for alterations in the entire mitochondrial genome with restriction enzymes that have several polymorphic restriction sites in the genome.