A Highly Specific Microarray Method for Point Mutation Detection

Abstract

Improvements of microarray techniques for genotyping purposes have focused on increasing the reliability of this method. Here we report the development of a genotyping method where a microarray was spotted with stemloop probes, especially designed to optimize the hybridization specificity of complementary DNA sequences. This accurate method was used to screen for four common disease-causing mutations involved in a neurological disorder called Charcot-Marie-Tooth disease (CMT). Healthy individuals' and patients' DNA were amplified and labeled by PCR and hybridized on microarray. The spot signal intensities were 81 to 408 times greater for perfect compared with mismatched target sequences, differing by only one nucleotide (discrimination ratio) for healthy individual "homozygous" DNA. On the other hand, "heterozygous" mutant DNA samples gave rise to signal intensity ratios close to 1, as expected. The genotypes obtained by this method were perfectly consistent with those determined by direct PCR sequencing. Cross-hybridization rates were very low, resulting in further multiplexing improvements. In this study, we also demonstrated the feasibility of real-time hybridization detection of labeled synthetic oligonucleotides with concentrations as low as 2.5 nM.

Real-time DNA microarrays: reality check

“…Although rational probe design is a necessary tool in any hybridization experiment, it cannot solve the problem of multiplexing in general cases. For example, by designing hairpin probes, we can theoretically achieve higher specificity of target recognition; however, in practice, this effect can be negated by differences in target concentrations in the sample [43]. …”

Fast development of CE IVD molecular diagnostic kits: Highly specific microarray kit for 49 cystic fibrosis mutations

“…The HairLoop TM probe performances were assessed elsewhere [13] by hybridizing mixes of synthetic oligonucleotides matching the wild type or mutant alleles. Hairloop TM probe hybridization specificity was determined by measuring the cross hybridization rate and the discrimination ratio for each probe.…”

“…Recently, significant progress has been made in low-density microarrays having below 400 spots/cm 2 in order to eliminate limitations regarding specificity [13], cost, or amount of work needed for detection of mutations or sequences. Several methods for single nucleotide polymorphisms (SNP) genotyping with DNA microarrays have been described.…”

Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

“…Our probes, also called hairloops, consist of a GC-rich stem and a target-specific loop [15]. After hybridization with perfectly complementary labeled targets, the stems of probes are opened for the subsequent detection of these targets.…”