title: ITB UniversalArray 49 v2
status: Public on Sep 26 2013
contact.city: Bologna
contact.first: Simone
contact.last: Rampelli
contact.email: simone.rampelli@unibo.it
contact.address: Via Belmeloro 6
contact.country: Italy
contact.institute: University of Bologna
organism: synthetic construct
description: The Ligase Detection Reaction (LDR) technology of HTF-Microbi.array is based on the discriminative properties of the DNA ligation enzyme. This technique requires the design of a pair of two adjacent oligonucleotides specific for each target sequence: a probe specific for the variation (called "Discriminating Probe", or DS) which carries a 5'-fluorescent label, and a second probe, named "Common Probe" (or CP), starting one base 3'-downstream of the DS that carries a 5'-phosphate group and a unique sequence named cZipCode at its 3'-end. The oligonucleotide probe pairs and a thermostable DNA ligase are used in a LDR reaction with previously PCR-amplified DNA fragments. The LDR products, obtained only in presence of a perfectly matching template by action of the DNA ligase, are addressed to a precise location onto a Universal Array (UA), where a set of artificial sequences, called Zip-codes are arranged. These products carry both the fluorescent label and a unique cZipCode sequence and can be detected by laser scanning and identified according to their location within the array.
  
  Refrence - Candela M, Consolandi C, Severgnini M, Biagi E, Castiglioni B, et al. High taxonomic level fingerprint of the human intestinal microbiota by ligase detection reaction--universal array approach. BMC Microbiol. 2010 Apr 19;10:116. doi: 10.1186/1471-2180-10-116.
manufacture_protocol: Phenylen-diisothiocyanate activated chitosan glass slides were used as surfaces for the preparation of universal arrays, comprising a total of 49 Zip-codes. In particular, glass slides were cleaned by soaking in 1M NaOH for 2 h followed by rinsing with distilled water, immersed in 1N HCl solution overnight a shaker, and then rinsed again in distilled water. Slides were immersed in 96% ethanol for 10 min and then washed three times with distilled water. Slides were immersed in acetone for 10 min, removed, and dried. Then they were treated with 1% (v/v) GOPS (3-glycidoxypropylsilane) in 95% ethanol for 1 h. Excess silane was removed by dipping the slides in 95% ethanol for 1 min. Finally they were dried at 150 °C for 20 min. The polymer solution was prepared by dissolving 10 g of chitosan (400 kDa) in a liter of 50 mM HCl. The solutions were heated at 70 °C, stirred for 1 h, and then filtered. Treated slides were left in a water solution containing 10% PBS and 10% polymer (0.1% w/v) for 1 h on a shaker. The slides were washed five times with distilled water, centrifuged at 800 rpm for 3 min. and dried for 10 min at 45 °C. The slides were then activated by immersion into a 0.2% solution of 1,4-phenylene diisothiocyanate in 90% N,N-dimethylformamide and 10% pyridine. The activation reaction was carried out at room temperature for 2 h. After being washed with methanol and acetone (2 min each), the activated slides were dried and stored, until used, in a dark closed box under vacuum. Solutions of 5‘-amino-modified oligonucleotides with a 5‘-polyA tail, called zip-codes, were transferred into a 384-well microtiter plate in a volume of 30 μL and spotted at a final concentration of 50 μM in 150 mM sodium phosphate buffer (pH 8.5). The spotting was performed using a contact system (MicroGrid II Compact, Biorobotics, UK).