title: Chromochip Arabidopsis thaliana 21.7K CHROMO4_1
status: Public on Sep 10 2010
contact.city: Gif sur Yvette
contact.first: Véronique
contact.last: BRUNAUD
contact.email: veronique.brunaud@inrae.fr
contact.address: avenue des sciences
contact.country: France
contact.institute: INRAE
organism: Arabidopsis thaliana
description: The entire TIGR4 Arabidopsis thaliana ec. Col chromosome 4 assembly was used to design a genomic tiling microarray. This ~18.6 Mb sequence represented on the microarray starts downstream of the nucleolar organizing region (NOR) comprising the 5’ terminus of the chromosome. Notably, it contains several megabases of pericentromeric heterochromatin. In the microarray design, annotation was intentionally ignored, thus ensuring that repeated and unique sequences were equally represented. PCR primer pairs were selected using the Primer3 software. Probe selection was first accomplished by BLASTN analysis of sequential 100-bp windows of sequence along chromosome 4 against the whole genome to discriminate between unique and repeated DNA. Whenever a window gave at least one hit with 85% identity elsewhere in the genome, that window was marked as repeated, or unique otherwise. This information was then used to define repeated and unique segments of at least 300bp along the chromosome, as follows. Repeat segments were composed of successive repeat windows, possibly interrupted by series of contiguous unique windows spanning less than 300bp in each case. Conversely, unique segments were composed of successive unique windows, never interrupted by more than 300bp of contiguous repeat windows. Series of alternate unique and repeated windows were treated as repeat segments. Within each repeated or unique segment, PCR primer pairs were then selected using the Primer3 software and a routine that ensured maximum coverage of the segment and limited overlap between successive probes. Briefly, primer pairs were designed from both ends of the segment, considering sequential 1.2 kb windows, amplicon sizes ranging from 850 bp to 1.2 kb (optimum 1 kb) and primers (19-23 nt each, optimum 21 nt, 40-60% GC) with similar melting temperatures in the 57°C­63°C range (optimum 60°C). Each new 1.2 kb window was positioned 50 bp within the end of the preceding amplicon. Successive amplicons could therefore overlap by up to 50 bp or be separated by up to 250 bp. When no primer pair was found in a given window, window size was increased to 1.4 kb, and if still unsuccessful, amplicons were finally designed using the forward and reverse primers of the upstream and downstream amplicons, respectively. When the last (central) window of a segment was less than 1.2kb but more than 300bp, primer pair selection was carried out by considering the actual size of that window plus 50bp on either side. Using this approach, 21,405 amplicons were designed with an average size of ~950bp. An additional 356 amplicons were designed that cover 36 genes of interest and neighboring sequences located on the other 4 chromosomes. Most amplicons were produced using BAC clones as templates, and all amplicons were verified by gel electropheresis.
manufacture_protocol: http://urgv.evry.inra.fr/CATdb