title: Custom Theileria parva oligonucleotide microarray
status: Public on Jul 30 2009
contact.city: New York
contact.first: Simon
contact.last: Kang'a
contact.state: NY
contact.address: 341 East 25th Street
contact.department: Medical Parasitology
contact.country: USA
contact.institute: New York University School of Medicine
organism: Theileria parva strain Muguga
manufacture_protocol: Microarray design and printing was performed by Dr. Simon Kang’a at The Institute for Genomic Research (TIGR), now the J. Craig Venter Institute (JCVI). A total of 4060 unique 70 mer oligonucleotides were designed from an initial 4082 predicted ORF of the Theileria parva (Muguga) genome using the Pick70 program of ArrayOligoSelector software (http://arrayoligosel.sourceforge.net). Oligonucleotide design considerations included uniqueness, avoidance of internal self-annealing structures, narrow annealing temperature (Tm) range over the entire oligonucleotide set, masking of low-complexity regions, balanced base composition (28-30%GC) and proximity to the 3' end. The TIGR genome sequence database containing contigs of the entire T. parva genome was used as the “complete genome source” for screening the 70-mer oligonucleotide sequences for uniqueness using the BLAT feature as implemented in Pick70. The T. parva 70-mer oligonucleotides were also compared by BLAST search against bovine sequences in GenBank and TIGR bovine Gene Index to ensure that they did not cross-hybridize with any bovine host DNA. The 70-mer oligonucleotides were purchased from Illumina (Illumina Inc., San Diego, CA) at a concentration of 50 μM, diluted to 25 μM using an equal volume of Corning Universal Spotting buffer (Corning Inc, Corning, NY) and spotted in quadruplicate onto Corning® UltraGAPS™ aminosilane coated slides (Corning Inc, Corning, NY) using an Intelligent Automation Systems (IAS) arrayer (Cambridge, MA) with a 48-pen print head configuration (12 rows by 4 columns). After printing, the spotted oligonucleotide DNA was cross-linked to the slide surface by UV irradiation at an integrated intensity of 1200 mJ cm-2 using a Stratalink UV Crosslinker (Stratagene, La Jolla, CA). The slides were stored in a desiccator chamber until use.