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Biomedical subjects

Meiju Ji

Publications and source records attributed to Meiju Ji.

13 recordsLinked to original sources
PubMed2006

Exo-Dye-based assay for rapid, inexpensive, and sensitive detection of DNA-binding proteins.

We reported herein a rapid, inexpensive, and sensitive technique for detecting sequence-specific DNA-binding proteins. In this technique, the common exonuclease III (ExoIII) footprinting assay is coupled with simple SYBR Green I staining for monitoring the activities of DNA-binding proteins. We named this technique as ExoIII-Dye-based assay. In this assay, a duplex probe was designed to detect DNA-binding protein. One side of the probe contains one protein-binding site, and another side of it contains five protruding bases at 3' end for protection from ExoIII digestion. If a target protein is present, it will bind to binding sites of probe and produce a physical hindrance to ExoIII, which protects the duplex probe from digestion of ExoIII. SYBR Green I will bind to probe, which results in high fluorescence intensity. On the contrary, in the absence of the target protein, the naked duplex probe will be degraded by ExoIII. SYBR Green I will be released, which results in a low fluorescence intensity. In this study, we employed this technique to successfully detect transcription factor NF-kappaB in crude cell extracts. Moreover, it could also be used to evaluate the binding affinity of NF-kappaB. This technique has therefore wide potential application in research, medical diagnosis, and drug discovery.

Amino Acid Sequence↗
PubMed2006

Quantitative analysis of promoter hypermethylation in multiple genes in osteosarcoma.

BACKGROUND: Osteosarcoma is the most common solid malignant diseases of childhood, occurring in approximately 6 children per million annually; however, to the authors' knowledge to date, the cause of osteosarcoma has remained mostly unknown. Genetic alterations of genes that are specific for osteosarcoma have not been identified. Genetic alternations in the status of DNA methylation, known as epigenetic alterations, are the most common molecular alterations in human neoplasia. Aberrant methylation in the promoter region of tumor-related genes is associated closely with epigenetically mediated gene silencing, which is a common feature in human tumors. METHODS: The authors analyzed CpG islands of 5 different gene loci for aberrant methylation profiles in 30 pairs of osteosarcoma and corresponding normal tissues by using the quantitative methylation-specific polymerase chain reaction method. The objectives of this study were to characterize the methylation changes in osteosarcoma more extensively and to identify epigenetic biomarkers that may be useful in the diagnosis and prevention of osteosarcoma. RESULTS: For the Ras effector homologue (RASSF1A), tissue inhibitor of metalloproteinase 3 (TIMP3), O-6-methylguanine DNA methyltransferase (MGMT), and death-associated protein kinase 1 (DAPK1) genes, significant differences were observed in the degree of hypermethylation between tumors and normal tissues (P < 0.01 and P < 0.001, respectively). Measurement of the cumulative multiple promoter hypermethylation revealed striking differences between tumor specimens and normal tissues (t = 7.31; P < .001). There also was a significant difference in the levels of DNA methylation between the metastatic and nonmetastatic high-grade osteosarcomas (t = 4.57; P < .01). In addition, the methylation levels were associated closely with gender (t = 6.44; P < .001). CONCLUSIONS: The results indicated that tumor tissues from patients with osteosarcoma had a significantly higher incidence of hypermethylation for several genes compared with corresponding normal tissues. The epigenetic changes observed in this study may have prognostic importance for patients with osteosarcoma.

Adolescent↗
PubMed2005

Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of p16 INK4A gene.

A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ( INK4A ) gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ( INK4A ) gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.

Base Pair Mismatch↗
PubMed2004

High-throughput method for detecting DNA methylation.

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.

Automation↗
PubMed2004

Microarray-based method for genotyping of functional single nucleotide polymorphisms using dual-color fluorescence hybridization.

Screening disease-related single nucleotide polymorphism (SNP) markers in the whole genome has great potential in complex disease genetics and pharmacogenetics researches. It has led to a requirement for high-throughput genotyping platforms that can maximize the efficient screening functional SNPs with respect to accuracy, speed and cost. In this study, we attempted to develop a microarray-based method for scoring a number of genomic DNA in parallel for one or more molecular markers on a glass slide. Two SNP markers localized to the methylenetetrahydrofolate reductase gene (MTHFR) were selected as the investigated targets. Amplified PCR products from nine genomic DNA specimens were spotted and immobilized onto a poly-l-lysine coated glass slide to fabricate a microarray, then interrogated by hybridization with dual-color probes to determine the SNP genotype of each sample. The results indicated that the microarray-based method could determine the genotype of 677 and 1298 MTHFR polymorphisms. Sequencing was performed to validate these results. Our experiments successfully demonstrate that PCR products subjected to dual-color hybridization on a microarray could be applied as a useful and a high-throughput tool to analyze molecular markers.

Fluorescent Dyes↗
PubMed2004

Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI.

A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.

Base Pair Mismatch↗
PubMed2004

Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR.

BACKGROUND: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. Recently, several DNA microarray methods have been developed to detect the methylation status of the multiple genes. However, the microarrays are currently detected in fluorescence using a sophisticated laser-based scanner. These methods are limited by their lower sensitivity. METHODS: We present a sensitive colorimetric silver method coupled with linker-PCR to detect methylation status of four genes, including p16, E-cadherin, VHL, and hMLH1. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin used to detect biotinylated DNA. RESULTS: We show that p16, E-cadherin, VHL, and hMLH1 were all methylated in the positive control. However, no methylation was found in these genes for the negative control. P16 gene was only methylated in the sample, whereas others were not methylated. Furthermore, as little as 0.1 fmol of target DNA amplicons could be detected on the arrays. The results were further validated by methylation-specific PCR (MSP) and bisulfite DNA sequencing. CONCLUSIONS: The colorimetric silver detection of DNA microarray could be used as an inexpensive, useful, and sensitive tool to detect methylation status of the multiple genes in the future.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Covalent immobilization of DNA onto functionalized mica for atomic force microscopy.

The immobilization of DNA on the self-assembled monolayer of 3-aminopropyltrimethoxysilane (APTES) on mica wafer functionalized with glutaraldehyde (GA) by chemical bonding was studied by atomic force microscopy (AFM). The DNA used for our investigation was amplified by polymerase chain reaction, and primers were labeled with a -NH2 group at their 5' terminus. The surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and AFM. Results from XPS and AFM showed that the mica with the APTES and activated with GA can be formed, and the flatness of the mica can be adapted for AFM images. We found that the modified surface was capable of binding DNA molecules so that it withstood a thorough rinsing with a solution of sodium dodecylsulfate. Covalent binding between the aldehyde-terminated membrane and -NH2 groups at both ends of double-stranded DNA resulted in immobilization and straightening of the DNA.

Aluminum Silicates↗
PubMed2003

Detection of methylation of human p16(Ink4a) gene 5'-CpG islands by electrochemical method coupled with linker-PCR.

Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an electrochemical procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the coupling of DNA electrochemical sensors with linker-PCR- amplified DNA from human gastric tumor tissue and whole blood cells of healthy human. The synthesized oligonucleotide was immobilized on the modified gold electrode to fabricate a DNA biosensor. The hybridization reaction on the electrode surface was monitored by cyclic voltammogram (CV) and square wave voltammogram (SWV), using [Co(phen)(3)](ClO(4))(3) as a redox indicator. Methylation status of human p16(Ink4a) gene was detected and the results were validated by bisulfite DNA sequencing. A good reproducibility was observed in several parallel experiments. The coupling of DNA electrochemical sensors with PCR allowed quick detection and have the potential of the quantitative evaluation of the methylation status of the human p16(Ink4a) gene.

Base Sequence↗
PubMed2003

A microarray to analyze methylation patterns of p16(Ink4a) gene 5'-CpG islands.

OBJECTIVES: Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation on the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput approach for analyzing methylation patterns. The aim of this study was to develop a new method to analyze methylation patterns of p16(Ink4a) gene. DESIGN AND METHODS: We selected a 336 bp segment of the 5' untranslated region and the first exon of the p16(Ink4a) gene, as the target sequence, which include the most densely packed CpG fragment of the islands containing 32 CpG sites. A set of oligonucleotide probes was designed to assemble a DNA microarray to discriminate the methylation patterns of several adjacent CpG sites. RESULTS: Methylation patterns of human p16(Ink4a) gene were mapped and the results were validated by bisulphite DNA sequencing. A good reproducibility was observed in several parallel experiments. CONCLUSIONS: The methylation oligonucleotide microarray can be applied as a useful and powerful tool to map methylation patterns in multiple CpG island sites.

Base Sequence↗
PubMed2002

[Present status and prospects in research on effect of enhanced UV-B radiation on plants].

Current research status on the effect of enhanced UV-B radiation on plants was reviewed in this paper. The main research fields involved plant growth and morphological structure, plant physiological and biochemical metabolism, plant genetic material, UV-B-absorbing compounds and some gene expression, population, and ecosystem. In addition, some issues worthy to be studied in this field in future were proposed.

Ecosystem↗