title: Construction of a multiplex allele-specific PCR-based universal array (ASPUA) and its application to hearing loss screening.
abstract: We demonstrate a new method, using a universal array approach termed multiplex allele-specific PCR-based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele-specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele-specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin-coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost-effective, and easily-used, and is especially appropriate for mutation detection in clinical genetic diagnostics.
authors.affiliation: Medical Systems Biology Research Center, School of Medicine, Tsinghua University, Beijing, China.
authors.forename: Cai-Xia
authors.initials: CX
authors.lastname: Li
authors.forename: Qian
authors.initials: Q
authors.lastname: Pan
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Guo
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Hua-Fang
authors.initials: HF
authors.lastname: Gao
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.forename: Hao
authors.initials: H
authors.lastname: Hu
authors.forename: Wan-Li
authors.initials: WL
authors.lastname: Xing
authors.forename: Keith
authors.initials: K
authors.lastname: Mitchelson
authors.forename: Kun
authors.initials: K
authors.lastname: Xia
authors.forename: Pu
authors.initials: P
authors.lastname: Dai
authors.forename: Jing
authors.initials: J
authors.lastname: Cheng
mesh_terms: D000483:Alleles; D000072259:Connexin 26; D017630:Connexins; D034381:Hearing Loss / Q000175:diagnosis*; D006801:Humans; D008027:Light; D008403:Mass Screening; D046228:Microarray Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26