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

Abstract

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.

Microfluidic Platform for Single Nucleotide Polymorphism Genotyping of the Thiopurine S-Methyltransferase Gene to Evaluate Risk for Adverse Drug Events

“…RFLP, which has been implemented on existing microfluidic platforms, 24,31 uses restriction enzymes to digest DNA at specific sequences in an amplicon flanking the SNP of interest. FRRFLP is widely used to perform SNP genotyping, and in computational models is applicable to up to 85% of SNPs in the National Center for Biotechnology Information database, (dbSNP).…”

“…18 -20 Although there are numerous methods to genotype SNPs, 21 most require substantive infrastructure, highly trained operators, and batch processing of many samples. Several microchip-based techniques have been demonstrated for mutation detection, but most involve large-scale genotyping or mutational screening [22][23][24][25][26][27][28] and some off-chip processing. Work to date 1,[3][4][5] has primarily used expensive glass microchips in which mainly capillary electrophoresis (CE)-based functionality is included.…”

High resolution DNA separations using microchip electrophoresis

“…Diverging from the use of a combined HDA and SSCP approach, an integrated on-chip HDA/RFLP methodology was reported; the discrimination of both heterozygous and homozygous mutations present in the HFE gene using microchip electrophoresis was shown, which the HDA method alone was incapable of detecting [21].…”

An integrated method for mutation detection using on‐chip sample preparation, single‐stranded conformation polymorphism, and heteroduplex analysis

“…We recently presented a method of microchip HA that is much simpler to implement than others developed to date [22] while providing performance comparable to that of DHPLC. More recently [25], we combined HA and restriction-length polymorphism analysis, (i.e., sizing and sequence-specific enzymatic cleavage of DNA) to increase the sensitivity of the method. Jin et al [26] and Andersen et al [27] have also presented reviews of microchip-based mutation detection methods.…”

“…Following PCR, the samples were stored frozen at -207C. Further details can be found in [22] and [25]. The primers were not fluorescently labeled and were not designed specially for this application -they bracket the desired mutation, and do not amplify homologues.…”