A highly specific microarray method for point mutation detection

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Biotechniques. 2008 Jan;44(1):119-26.

A highly specific microarray method for point mutation detection.

Baaj Y, Magdelaine C, Ubertelli V, Valat C, Talini L, Soussaline F,

Khomyakova E, Funalot B, Vallat JM, Sturtz FG.

Department of Biochemistry and Molecular Genetics, University of

Limoges, Paris.

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.