We report a method called SSH array which combines the suppression subtraction hybridization (SSH) and DNA array techniques to find species specific DNA probes from genomic DNA (gDNA) for species identification. The method first obtains the differential gDNA fragments between two species by SSH and then hybridizes the differential gDNA fragments with arrays made of multiple whole genomes from several species to screen the unique gDNA fragments for one species. The screened unique gDNA fragments can be used as species specific probes to differentiate the species they represent from all other species. We used five species of the genus Dendrobrium, D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., as experimental materials to study the feasibility of the method. The results showed that the method could efficiently obtain different species-specific probes for each of the five species.

We describe a new method for the assay of sequencespecific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNAfootprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNAbinding proteins. We named this method as exonuclease III(ExoIII) protectionassaywithFRETprobe.The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion,resulting in high FRET. However, if the target protein does not exist, theFRETpaironthe nakedFRETprobe will be degraded by ExoIII, resulting in low FRET.Three kinds of recombinant transcription factors including NF-kB, SP1 and p50, and the target protein of NF-kB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3Vhydroxyl end and an overhang sequence at 5Vamino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3V end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-nB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.

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 5V end were firstly annealed to a same universal oligonucleotide with amino group at 5V 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 3V 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. D 2005 Elsevier B.V. All rights reserved.

This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor κB (NF-κB). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT, contained a NF-κB binding consensus sequence for capturing activated NF-κB in analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-κB. Finally, the probes protected by NF-κB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-κB without the need for NF-κB antibodies. EMEA may provide a general approach for assays of DNA sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.