玻璃表面cDNA微阵列的制备与优化

THE FABRICATION AND OPTIMIZATION OF cDNA ARRAYSON THE GLASS SURFACE

对DNA进行荧光标记,确定简单、实用和有效的固定化方法和筛选出合适的杂交条件 ,是制备DNA阵列的几个关键步骤。在PCR的过程对cDNA进行标记 ,不同的掺入比例可以得到不同的标记效率,Cyedye -dCTP/dCTP的比例在1:3～1:4较好。将DNA固定到玻璃表面 ,poly -l-lysine方法是一种不需要对DNA进行修饰的有效方法。其中UV照射的能量以600mJ效果最好 ;水合(rehydrate)温浴过程的温度以65°C最佳。在固化过程中所用探针的浓度0.2 -0.5mg/ml对杂交信号影响不多 ,也就是芯片表面固定的探针DNA应选在此范围内。杂交信号与所用的靶DNA的浓度成正相关 ,且在一定的范围内呈正比例关系。

DNA was labeled with Cy3 or Cy5 during process of PCR. The optimized ratio of Cy3-dCTP/dCTP is between 1:3 and 1:4. Coupling strategy was currently used to graft DNA onto a glass surface by poly-l-lysine. The method involves coating a glass surface with a layer of poly-l-lysine and then spotting unmodified double-stranded DNA onto the coated glass in high salt to promote adsorption DNA to the poly-l-lysine layer. Both the coupling procedure and the hybridization efficiency have been optimized. The optimized temperature is 65℃. The optimized UV energy is 600mJ (BioRad UV crosslinker). The optimized concentration of probe DNA is 0.2mg/ml to 0.5mg/ml.