Testing and improving experimental parameters for the use of low molecular weight targets in array-CGH experiments.
Array-comparative genomic hybridization (CGH) has evolved as a useful technique for the detection and characterization of deletions, and, to a lesser extent, of duplications. The resolution of the technique is dictated by the genomic distance between targets spotted on the microarray, and by the targets' sizes. The use of region-specific, high-resolution microarrays is a specific goal when studying regions that are prone to rearrangements, such as those involved in deletion syndromes. The aim of the present study was to evaluate the best experimental conditions to be used for array-CGH analysis using low molecular weight (LMW) targets. The parameters tested were: the target concentration, the way LMW targets are prepared (either as linearized plasmids or as purified PCR products), and the way the targets are attached to the array-CGH slide (in a random fashion on amino-silane coated slides, or by one amino-modified end on epoxysilane-coated slides). As a test case, we constructed a microarray harboring LMW targets located in the CREBBP gene, mutations of which cause the Rubinstein-Taybi syndrome (RTS). From 10 to 15% of RTS patients have a CREBBP deletion. We showed that aminosilane- and epoxysilane-coated slides were equally efficient with targets above 1,000 bp in size. On the other hand, with the smallest targets, especially those below 500 bp, epoxysilane-coated slides were superior to aminosilane-coated slides, which did not allow deletion detection. Use of the high resolution array allowed us to map intragenic breakpoints with precision and to identify a very small deletion and a duplication that were not detected by the currently available techniques for finding CREBBP deletions.
Stef
Marianne
M
Laboratoire de Génétique Humaine, Développement et Cancer (Equipe d'Accueil 3669), Université Victor Segalen Bordeaux2, Bordeaux, France.
Simon
Delphine
D
Burgelin
Ingrid
I
Guisle
Isabelle
I
Chevalier
Catherine
C
Delrue
Marie-Ange
MA
Lacombe
Didier
D
Léger
Jean
J
Arveiler
Benoît
B
D015415:Biomarkers / Q000032:analysis*; D050882:CREB-Binding Protein / Q000737:chemistry / Q000235:genetics; D019457:Chromosome Breakage; D004252:DNA Mutational Analysis / Q000379:methods; D004852:Epoxy Compounds / Q000737:chemistry; D017353:Gene Deletion; D020440:Gene Duplication; D005820:Genetic Testing / Q000379:methods*; D023281:Genomics / Q000379:methods; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012107:Research Design; D012821:Silanes / Q000737:chemistry; D013501:Surface-Active Agents / Q000032:analysis
D015415:Biomarkers; D004852:Epoxy Compounds; D012821:Silanes; D013501:Surface-Active Agents; D050882:CREB-Binding Protein; C494363:CREBBP protein, human