Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis

This work describes an integrated method of enzymatic digestion, heteroduplex analysis (HA) and electrophoretic sizing on a microfluidic chip. HA techniques based on microchip electrophoresis are capable of the high sensitivity detection of subtle mutations such as single nucleotide polymorphisms (SNPs) but are not readily able to detect homozygous mutant genotypes. Such homozygous conditions are commonly encountered with the gene implicated in hereditary haemochromatosis, HFE. We employed the restriction fragment length polymorphism (RFLP) method of mutation detection to complement the HA method in a rapid novel on-chip procedure that separated digested PCR fragments to reliably determine the presence or absence of the most important mutations associated with haemochromatosis. This method was able to distinguish the homozygous mutant, heterozygous and homozygous wildtype genotypes. The mutations investigated here (C282Y, H63D and S65C) are often the mutation targets used in the genetic testing for haemochromatosis. This method provides the extremely specific digestion methods needed for the analysis of the known and relatively common mutations that have a significant probability of occurring in a homozygous form. However, the high sensitivity of the HA method is useful in detecting other mutations of lesser likelihood which, by virtue of their rarity, are likely to be present only in a heterozygous form. Although the conventional methods of analysing these mutations require as much as a day to perform, this microchip method, even without robotics or multiplexed operation, can be performed in about 10 min per sample.