Optimization of on-chip elongation for fabricating double-stranded DNA microarrays.
The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiments showed that the elongation temperature of 50 degrees C and the Mg(2+) concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.
Bai
Yunfei
Y
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Ge
Qinyu
Q
Wang
Jinke
J
Li
Tongxiang
T
Liu
Quanjun
Q
Lu
Zuhong
Z
D002232:Carbocyanines; D004247:DNA; D004256:DNA Polymerase I; D005898:Glass; D005976:Glutaral; D015636:Magnesium Chloride; D016328:NF-kappa B; D020411:Oligonucleotide Array Sequence Analysis; D019914:Taq Polymerase; D013696:Temperature
D002232:Carbocyanines; D016328:NF-kappa B; C089187:cyanine dye 3; D015636:Magnesium Chloride; D004247:DNA; D019914:Taq Polymerase; D004256:DNA Polymerase I; D005976:Glutaral