Zu H Lu | PubMed Health

PubMed2005

A method for fabricating uni-dsDNA microarray chip for analyzing DNA-binding proteins.

This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5' end were firstly annealed to a same universal oligonucleotide with amino group at 5' end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3' end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins.

Cost-Benefit Analysis↗

PubMed2003

Evaluating the binding affinities of NF-kappaB p50 homodimer to the wild-type and single-nucleotide mutant Ig-kappaB sites by the unimolecular dsDNA microarray.

This study investigated the binding affinities of NF-kappaB p50 homodimer to the wild-type and single-nucleotide mutant Ig-kappaB sites by the unimolecular dsDNA microarray which was fabricated with a novel scheme. The importance of each nucleotide of Ig-kappaB site for the sequence-specific p50p50/Ig-kappaB interaction was thus evaluated. The results demonstrate that the nucleotides at different positions contribute differently to the p50p50/Ig-kappaB binding interaction. The G(1), G(2), and C(10) are most important for p50p50/Ig-kappaB binding interaction and determine the specificity of p50p50/Ig-kappaB interaction, which replacements with any other nucleotide could result in the similarly greatest binding affinity losses. Comparatively, the G(3), A(4), T(8), and C(9) are less important for p50p50/Ig-kappaB interaction and regulate the binding affinity, which substitutions with the variant nucleotide could change the binding affinity differently. The C(5) is least important for p50p50/Ig-kappaB interaction, the randomized nucleotide exchange of which little affects on p50p50/Ig-kappaB binding affinity. Among all possible single-nucleotide mutants, the T(8) to C mutation could strengthen p50p50/Ig-kappaB interaction. The T(7) acts differently from its symmetric C(5) and the axial T(6) is necessary for high-affinity p50p50/Ig-kappaB interaction. The unimolecular dsDNA microarray provides a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.

Binding Sites↗