Biomedical subjects

Tongxiang Li

Publications and source records attributed to Tongxiang Li.

6 recordsLinked to original sources

PubMed2006

Evaluating the binding affinities of NF-kappaB protein to the single-nucleotide mismatch DNA binding sites by using double-stranded DNA microarray.

Protein-DNA sequence-specific interaction plays an essential role in many biological processes. Here we immobilized a series of double-stranded DNA probes on an agarose coated slide to investigate the binding affinity of NF-kappaB p50 homodimer to the single-nucleotide mismatches (G<-->A or T<-->C) of the 10 base pair (bp) protein binding sites. The results demonstrated that the nucleotides at different positions contribute differently to the p50p50/DNA binding interaction. Within the 10 bp binding sites, the 5tG or 6cA mismatch has less effect on the protein-DNA binding affinity. Even the 5tG mismatch may have the ability to enhance the protein-DNA interaction (5t/w = 1.07). On the other hand, the 7cA or 10tG mismatch blocked the protein-DNA interaction more significantly than other six single-nucleotide mismatches. (7c/W = 0.37, 10t/W = 0.35). It also indicated that the duplex DNA probes immobilized on the agarose-coated surface were apt to be recognized by DNA-binding proteins, and this method would provide a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.

Base Pair Mismatch↗

PubMed2005

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.

Carbocyanines↗

PubMed2005

Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins.

We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. 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, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-kappaB, SP1 and p50, and the target protein of NF-kappaB 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.

Cell Nucleus↗

PubMed2005

A free-labeled method for DNA-binding protein detection using a double-stranded DNA microarray.

We have developed a new type of double-stranded DNA microarray to perform detection of sequence-specific DNA-binding proteins. The DNA-binding site of a DNA-binding protein is divided into two fragments. One fragment was immobilized on an aldehyde-coated glass microscope slide surface via chemical bonds. The other fragment was labeled with a fluorescent molecule. When using this kind of double-stranded DNA microarray, the labeled DNA fragment was pre-incubated with detection sample for 5 to 10 minutes and then hybridized with the microarray. In our experiment, six different concentrations of Nuclear Factor kappa-B P50 homodimer in detection samples were tested. The microarray fluorescence intensity was obtained and the relationship between the intensity and the protein concentration was calculated. The detection results suggested that this free-labeled detection system could have the ability to be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.

Base Sequence↗

PubMed2004

Accurate identification of closely related Dendrobium species with multiple species-specific gDNA probes.

About 63 species of Dendrobium are identified in China, making the identification of the origin of a particular Dendrobium species on the consumer market very difficult. We report evaluation of multiple species-specific probes screened from genomic DNA for closely related Dendrobium species identification, based on DNA array hybridization. Fourteen species-specific probes were screened from five closely related Dendrobium species, D. aurantiacum Kerr, D. officinale Kimura et Migo, D. nobile Lindl., D. chrysotoxum Lindl. and D. fimbriatum Hook., based on the SSH-Array technology we developed. Various commercial Dendrobium samples and unrelated samples were definitely identified. The specificity and accuracy of the multiple species-specific probes for species identification was assessed by identifying various commercial Dendrobium samples (Herba Dendrobii). Hybridization patterns of these multiple probes on digested genomic DNAs of Dendrobium species indicated that there are distinct polymorphic sequence fragment in the higher eukaryotes. This is the first report on detection and utilization of multiple species-specific probes of Dendrobium in whole genomic DNA, and this could be useful tools not only for a new technical platform for the closely related species identification but also for epidemiological studies on higher eukaryotes.

DNA↗

PubMed2003

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions.

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 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' 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-kappaB 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.

Binding Sites↗