RAPID-M™ technology: A cell-free method for protein microarray generation from PCR DNA

Abstract

IntroductionBurkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with multifarious manifestations and has been listed as a potential biowarfare agent. The successful management of patients with severe melioidosis depends on early diagnosis and initiation of appropriate antibiotic therapy. Protein microarray has shown enormous potential for in vitro diagnostics due to miniaturization whilst generating a maximum of diagnostically relevant information from minute amount of sample. The Repeatable Arrays of Proteins using Immobilized DNA Microplates (RAPID-MTM) technology was developed to facilitate the development of high-throughput protein microarray platform that potentially contributes to the diagnostic approach. ObjectiveTo develop protein microarray using RAPID-MTM technology for potential diagnostic applications MethodsThe Omp3 and GFP genes were immobilized onto beads during PCR amplification and these beads-immobilized DNA were then used as template for cell-free reactions. The synthesized proteins were printed directly onto glass slide using in situ protein purification to produce many identical copies of protein microarray. Result & DiscussionExpression of Omp3 and GFP proteins using beads-immobilized DNA yielded protein bands with expected molecular size of 27kDa and 30kDa, respectively as proven by the Western blot analysis. The Omp3 and GFP proteins were still functional after in situ purification on the Ni-NTA microarray slide. ConclusionProtein microarray fabrication using RAPID-MTM technology has been successfully developed.