[A novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology].

Abstract

Multiplex amplifiable probe hybridization (MAPH) has recently been developed to detect gene copy number changes in total genome in several genetic disease. Here we reported a novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology for DNA fragment copy number measurement. A set of amplifiable probes were prepared by locus-specific forward and reverse primers synthesized with the T7 and T3 promoter sites at their respective 5'-ends to the interest DNA fragments and purified by Qiagen PCR products purification kit. The set of probes were then hybridized with genome DNA immobilized on the nylon membrane. The selective probes after MAPH were collected with streptavidin coated magnetic beads and amplified by a biotin labeled universal primers. The biotinylated PCR products were then intended for hybridization to the corresponding oligonucleotide arrays consisted of 10 exons of DMD gene probes and positive,negative control probes immobilized on glass slides. After hybridization, the slides were washed by hybridization buffer and stained by streptavidin-Cy3. The hybridization fluorescence images were scanned by the ScanArray software and further quantitatively analyzed by the ImageJ software. Here we used oligonucleotide array technology to replace the agarose gel analysis for detecting the biotinylated PCR products so that extendable ability of parallel assay in our method had greatly been improved. One control female, one control male and one DMD patient were tested in this study. The results demonstrate the feasibility of high-throughput detection and relative quantification of DNA fragment copy numbers changes in total genome DNA combination of MAPH and high-density oligonucleotide arrays in single reaction.