一种基于寡核苷酸微阵列芯片的多重可扩增探针杂交技术

A Novel Oligonucleotide Arrays-based Multiplex Amplifiable Probe Hybridization Technology

多重可扩增探针杂交技术 (multiplexamplifiableprobehybridization ,MAPH)是近年来发展起来的一种用于基因组中DNA拷贝数检测的新技术。并发展了一种基于寡核苷酸微阵列芯片的MAPH技术。该方法根据所检测的DNA序列 ,制备若干具有通用引物的PCR产物作为可扩增探针组 ,与固定在尼龙膜上待测的基因组DNA杂交。用磁珠回收特异性杂交的探针 ,经生物素标记的通用引物扩增后 ,与相应的寡核苷酸微阵列芯片杂交。该特异性的寡核苷酸微阵列芯片包括 10个抗肌营养不良基因的外显子探针和阴性、阳性探针。杂交清冼后 ,链霉亲和素 Cy3染色用芯片扫描仪得到杂交的荧光图像。分析荧光信号的强度差异给出特定基因片段拷贝数的变化。该方法用微阵列技术代替MAPH中的电泳检测技术 ,可大幅度增加检测的通量。选择了一个正常男性、一个正常女性和一个肌营养不良症患者的基因组DNA来进行验证。结果表明 。

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.