Optimization of Hybridization Condition of 70 mer Oligonucleotide Probe Microarrays

Objective To optimize the conditions for the preparation,target gene labeling and hybridization of long oligonucleotide microarrays.Methods The probe concentrations of spotting and immobilization were optimized by direct labeling.The different labeling methods for target genes were compared,and the hybridization solutions and conditions were optimized.Results Sufficient signal intensity could be obtained when 70 mer oligonucleotide probe was used,and the probe concentration of spotting was 10 μmol/L.By hydration at 37 ℃ after spotting and then UV cross-linking at 3600 mJ,satisfied immobilization could be achieved.The optimized hybridization solution consisted of 50 % formamide,5×SSC and 0.5 %SDS.The optimal hybridization time was 8 hours and temperature was 65 ℃.The biotin indirect fluroscence labeling is more efficient and less expensive than the Cy3 direct labeling method for the target genes.Conclusion The optimal spotting concentration,target gene labeling and the hybridization solution and temperature were obtained and this lays the foundation for the development of oligonucelotide microarray for the parallel detection of different species of pathogen in human.