Biomedical subjects

Wen-li Ma

Publications and source records attributed to Wen-li Ma.

36 records · Page 2Linked to original sources

PubMed2004

[Co-expression of two spliceosomes of cyclin-dependent kinase-2 in SH-SY5Y cells].

OBJECTIVE: To investigate the expression of cyclin-dependent kinase-2 (CDK-2) gene in SH-SY5Y cells. METHODS: The expression of CDK-2 gene was examined with reverse transcriptional (RT)-PCR, and the PCR products underwent electrophoresis on non-denaturing poly-acrylamide gel (PAG) followed by silver staining. The separated and purified DNAs were ligated into pMD18-T vector, and the positive clones identified by sequence analysis. RESULTS: Two DNA bands were displayed on PAG, and the one with smaller molecular weight was less intensively stained. The sequences of the two clones indicated that both were products of CDK-2 gene. CONCLUSION: Two kinds of CDK-2 gene products are co-expressed in the SH-SY5Y cells, one of which lacks the fifth exon and is expressed at a low level.

CDC2-CDC28 Kinases↗

PubMed2004

[Application of microarrays in screening the tumor-specific genes in the genome of K562 cells].

OBJECTIVE: To screen tumor-specific genes of K562 cells using DNA microarray technique. METHODS: The genomic DNA of normal white blood cells and cultured K562 cells were respectively purified and digested with Sau3A I, and the digested DNA fragments of K562 cells were cloned into TA cloning vector to construct the corresponding genomic DNA library. The insert genomic DNA fragments were amplified from the library to prepare the microarray using Cartesian 5500 Microarrayer. The digested genomic DNA fragments of normal white blood cells were labeled with fluorescent Cy3 by restriction display PCR (RD-PCR), followed by hybridization with the microarray, after which the slide was washed and scanned with ScanArray. RESULTS: Among the 426 target genes, 42 differential genes were identified in the genomic DNA of K562 cells in comparison with the normal white blood cells. One of the genes was identified as the breakpoint cluster gene (BCR) after sequence analysis. CONCLUSIONS: The DNA microarrays we constructed may effectively identify the tumor-specific genes in K562 cells, and DNA microarray technique can be helpful in elucidating the molecular mechanisms of tumorigenesis at the genomic level.

Amino Acid Sequence↗

PubMed2004

[Isolation of the target gene from cDNA restriction fragments using 70-mer oligo microarray].

OBJECTIVE: To study the method for using a 70-mer oligo microarray as the probe to isolate target genes from the cDNA restriction fragments. METHOD: Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock culture and reversely transcribed into the double-stranded cDNAs, which were prepared into restriction cDNA fragments using restriction display (RD) method. The microarray was printed using a single 70-mer specific oligo designed to according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled by PCR method with the Cy5 universal primer before hybridization with the microarray. The microarray was stripped after washing and scanning, and the strip solution was collected for another round of PCR amplification using the universal primer without fluorescence. The PCR product was then cloned into PUC18 T vector and transformed into to E.coli JM109 cells for amplification, and the plasmids were extracted and sequenced for identification. RESULTS: BLAST results showed that the target gene was cloned successfully. CONCLUSION: The target gene can be isolated directly using the 70-mer oligo microarray as the probe from the cDNA fragments prepared by RD method, without the necessity of building a cDNA library. This method can also be used in further research to acquire the differentially expressed genes after the oligo microarray hybridization.

Base Sequence↗

PubMed2004

[Construction of phage antibody library for Fab fragment from a convalescent patient infected with SARS coronavirus].

AIM: To construct the phage antibody library for Fab fragment from a convalescent patient infected with SARS coronavirus. METHODS: Total RNA was extracted from peripheral blood lymphocytes of a patient with SARS virus-specific IgG antibody and was transcribed reversely into cDNA. Using the cDNA as template, Fd and light chain of Fab genes were amplified by PCR with specific primer and were cloned into phagemid pComb3. Then the recombinant phagemid was electroporated into E.coli XL-1 Blue. The recombinant rate was detected by enzyme digestion analysis, and library repertoire was determined. RESULTS: The antibody library of Fab fragment from a convalescent patient infected with SARS conronavirus was constructed. The recombinant rate of light chain and heavy chain of Fd gene were 91% and 75%, respectively. The library size was 7.23 x 10(7). CONCLUSION: Phage antibody library for human Fab fragment has been constructed successfully, which lays the foundation for further study.

Antibodies, Viral↗

PubMed2004

[An initial examination of the spermatozoal gene expression profile].

OBJECTIVE: To collect the normal spermatozoal gene expression sequence tags with the restriction display technique for constructing a microarray to understand spermatozoal gene expression profiles. METHODS: The total RNA extracted from normal human spermatozoa were reversely transcribed into cDNAs, which were digested by Sau3AI and linked to universal adapters (adapter 1) at both ends. According to the sequence of the adapter, a pair of primers (universal primers 1) was designed, followed by PCR with primers 1 and the PCR products were transferred into E.coli. The positive clones were collected after identification to serve as the probes for constructing the gene expression microarray of spermatozoa by printing those probes on the slides. The accomplished microarrays were examined by Cy3-labeled normal spermatozoal samples. RESULTS: Altogether 1 859 probes were collected, from which 368 were picked out randomly for constructing the microarray. CONCLUSIONS: Human spermatozoa contain a rich repertoire of RNAs, and the probes we prepared possess good incredibility and speciality.

Adult↗

PubMed2004

[A new method for screening mutant yeast strains with green fluorescent protein].

OBJECTIVE: To establish a fast and simple method for screening mutant yeast strains. MATERIALS AND METHODS: Homologous recombination technique was used to detect mutant yeast strains in yeast genomic library, with the green fluorescent protein gene as the reporter gene in the transposon. RESULTS: The strains that emitted green fluorescence were isolated, indicating that the gfp gene was inserted into the yeast genome by homologous recombination. CONCLUSION: This study established a useful method for functional genome study by homologous recombination technique, and provide an alternative for gene therapeutic drug development.

Cloning, Molecular↗

PubMed2004

[Oligonucleotide microarray preparation using enhanced poly-L-lysine glass slides].

OBJECTIVE: To modify conventional poly-L-lysine coating for oligonucleotide microarray preparation so as to enhance the sensitivity of the microarray. METHOD: The proposed chemical approaches included silanizing the slides with 3-glycid-oxypropyltrimethoxysilane (GOPS) after cleaning, followed by slide coating with polymers (poly-L-lysine) that was covalently bound to the modified glass. Subsequent attachment of the oligonucleotide to the modified slide surface was achieved after 1,4-phenylene diisothiocyanate (PDITC) activation of the surface. Various experiments were carried out, such as the immobilization efficiency and hybridization assays to test the modified slides, which were then used tentatively in the preparation of microarrays for SARS coronavirus detection. RESULTS: The improved surface had high immobilization efficiency, good uniformity and satisfactory hybridization efficiency, better than those slides with conventional poly-L-lysine coating. In addition, such modified slides rendered the microarrays more resistant to consecutive probing/stripping cycles. CONCLUSION: The modified slide surface is satisfactory to immobilize unmodified oligonucleotide by covalent binding, which enhances not only the sensitivity of the prepared oligonucleotide microarray but also the binding of the oligonucleotide to the slide surface.

DNA, Viral↗

PubMed2003

Possibility of using DNA chip technology for diagnosis of human papillomavirus.

To explore the application of DNA chip technology for the detection and typing of Human Papillomavirus (HPV), the HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by a PixSys 5500 microarrayer as probes to prepare the HPV gene chips. HPV samples, after being labeled with fluorescent dye by restriction display PCR (RD-PCR) technology, were hybridized with the microarray, which was followed by scanning and analysis. The experimental condition for preparing the HPV gene chips was investigated, and the possibility of HPV genotyping using gene chips was discussed. The technique that was established in this study for preparing HPV gene chips is practical. The results of the present study demonstrated the versatility and inspiring prospect of using this technology to detect and genotype HPV.

DNA Probes↗

PubMed2003

[Two restriction fluorescence labeling methods for enhancing the signal-to-noise ratio of cDNA microarray hybridization].

OBJECTIVE: To study the signal-to-noise ratio (SNR) of two restricted fluorescence labeling methods for examining gene expression profile by microarray hybridization. METHOD: Samples of Saccharomyces cerevisiae mRNA was labeled by traditional reverse transcription method and 2 restriction fluorescent labeling methods using respectively Cy-universal primer and extension incorporated Cy-dNTP. The labeled samples were examined by the microarray, followed by washing and scanning under the same conditions. RESULTS: The two restriction labeling methods showed superior results with lowered background and enhanced SNR and sensitivity, and Cy-universal primer labeling presented the best results. CONCLUSION: SNR can be enhanced by the restriction labeling methods, which improve the applicability of microarray technology.

Fluorescence↗

PubMed2003

Application of Agilent 2100 Bioanalyzer in detection of human papilloma virus.

OBJECTIVE: To explore the feasibility of using Agilent 2100 bioanalyzer in the detection and genotyping of human papilloma virus (HPV). METHODS: The consensus sequence of highly conserved region (L1) and genotype-specific gene fragments of all HPV genotypes (including HPV6, 11, 16 and 18) were amplified by PCR technique using general and type-specific primers respectively. Agilent 2100 Bioanalyzer and routine agarose gel electrophoresis were then employed respectively to examine the PCR products, and the accuracy, reproducibility and sensitivity of these 2 detection techniques were compared. RESULTS: Agilent 2100 Bioanalyzer showed better accuracy in determining the length of the gene fragments than agarose gel electrophoresis, the accuracy of the former reaching above 95% while the latter was only 85%. Bioanalyzer was also 100 times more sensitive than agarose electrophoresis. CONCLUSIONS: Agilent 2100 Bioanalyzer combined with PCR is specific, accurate and sensitive for HPV detection and genotyping.

Electrophoresis, Agar Gel↗

PubMed2003

Cloning and sequence analysis of Bacillus thuringiensis gene fragments isolated by restriction digest PCR.

OBJECTIVE: To clone and analyze Bacillus thuringiensis gene fragments isolated by restriction digest PCR (RD-PCR). METHOD: Specific primers were designed to amplify the genes of Bacillus thuringiensis israelensis (Bti), and the PCR products were classified and re-amplified by RD-PCR to obtain the fragments for subsequent purification and cloning into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. RESULTS: Sequence analysis showed that all the fragments amplified were Bti genes. CONCLUSION: RD-PCR is reliable in breaking down large gene fragments into confined and shorter gene fragments for preparing microarray probes.

Bacillus thuringiensis↗

PubMed2003

[Gene sequence analysis of SARS-associated coronavirus by nested RT-PCR].

OBJECTIVE: To explore an effective means for the detection of Severe Acute Respiratory Syndrome (SARS)- associated coronavirus. METHODS: The RNAs of the virus contained in the sputum samples from established SARS patients were extracted and reversely transcripted, followed by nested PCR using the reversely transcripted cDNA as the template. The PCR products were cloned then into the pMD18-T vectors, followed by sequence analysis. RESULTS: Specific fragments were amplified from the sputum samples of SARS patients, which were confirmed by DNA cloning and sequencing to belong to SARS-associated coronavirus. The Result of Blast shows only the difference in one nucleic acid from the TOR2 strain of SARS-associated coronavirus. CONCLUSION: Sequence analysis has confirmed the existence of SARS-associated coronavirus in the sputum samples of SARS patients, and nested RT-PCR is a quick, easy, and convenient way for the detection of the virus.

Base Sequence↗

PubMed2003

Rapid preparation of DNA microarray using PCR for hepatitis B and D virus detection.

OBJECTIVE: To prepare DNA microarray for detecting both hepatitis B and D virus as (HBV and HDV). METHODS: With the assistance of Oligo6.4 software, specific PCR primers targeting the conserved region of HBV and HDV were designed. The PCR products were purified and cloned into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were then extracted from positive clones and the target gene fragments underwent sequence analysis. RESULTS: The gene fragments of HBV and HDV were obtained by PCR, which were confirmed by sequence analysis to be specific gene fragments of HBV and HDV. CONCLUSION: Using PCR amplification products to prepare the DNA microarray is quick, simple and effective.

Hepatitis B virus↗

PubMed2003

[Preparation of gene chip probes for Bacillus anthracis protective antigen].

OBJECTIVE: To study the method for rapid preparation of the gene chip probes for Bacillus anthracis protective antigen (pag). METHODS: According to the sequences of pag published on PubMed, a pair of primers was designed to amplify the PA gene of Bacillus anthracis. Restriction enzyme digestion and AT clone were employed to rapidly screen out the recons of PA segments for the preparation of the gene chip probes. The DNA sequences of these segments were determined with 377 DNA Sequencer, and the resultant sequences analyzed with Bioinformatic software. RESULTS: With the designed primers, the pag 2,205 bp in length was amplified. After enzyme digestion and AT clone, 7 fragments of varied lengths were screened, which underwent analysis with the basic local alignment sequence tool (BLAST) and 377 DNA Sequencer. BLAST analysis showed that all the fragments cloned belonged to Bacillus anthracis. CONCLUSION: PCR in conjunction with restriction enzyme digestion and AT clone is rapid and simple to prepare the desired gene chip probes.

Antigens, Bacterial↗

PubMed2003

[Bioinformatic analysis of dengue virus cDNAs and design of oligonucleotide probes for microarray detection of the virus].

OBJECTIVE: To design oligonucleotide probes for microarray detection of dengue virus. METHODS: By analyzing the cDNAs of dengue viruses of 4 different serotypes with BLAST program, a group of specific sequences for the candidate probes was acquired. Oligo6.0 software was applied to analyze the candidates to select the probes with high specificity, identical length and similar melting temperature (Tm). RESULT: Altogether 48 oligonucleotide probes were designed, and deposited on oligonucleotide chips as the microarray for dengue virus detection. CONCLUSION: BLAST program and Oligo6.0 software are simple and effective means for designing the oligonucleotide probes.

Base Sequence↗

PubMed2003

Quick preparations of human parvovirus B19 microarray probes using PCR.

OBJECTIVE: To prepare DNA microarray probes for the detection of human parvovirus B19. Method Specific PCR primers were designed with the Primer Premier 5.0 to amplify the conserved regions of human parvovirus B19 genome. The PCR products were cloned into the pMD-18 T vector. METHOD: Sequences analysis showed the PCR products conformed to the sequences contained in the genome of human parvovirus B19. RESULT: Sequences analysis showed the PCR products conformed to the sequences contained in the genome of human parvovirus B19. CONCLUSION: PCR amplification of the conserved and specific human parvovirus B19 genes is simple and effective to prepare the desired probes.

DNA Probes↗

PubMed2002

[Analysis with DNA chips of the changes of gene expressions in K562 cells in response to As2O3 treatment].

OBJECTIVE: To investigate differential gene expression in apoptotic cells induced by As(2)O(3), and identify novel apoptosis-related genes. METHOD: Apoptosis of K562 cells cultured in RPMI 1640 medium supplemented with 10 % calf serum was induced by As(2)O(3). Total RNA of the apoptotic and normal cells were then extracted, purified and subject to reverse transcription into first-strand cDNA, labeled with Cy3/Cy5. Placenta DNA microarrays containing 348 DNA fragments were used to analyze the changes in gene expressions in the cells treated with As(2)O(3). RESULT: Eleven differentially expressed genes were identified in the apoptotic cells in comparison with the normal cells, 3 of which were associated with apoptosis, while the others were related to cell growth and proliferation. CONCLUSION: The placenta DNA microarrays we constructed may well apply to the analysis of the differentially expressed genes.

Antineoplastic Agents↗

PubMed2002

[Research advances on effect of arsenic trioxide on tumor].

Arsenic compounds are natural substances that have been used medically for more than 2,400 years in China. Since 1990s, Chinese physicians have began to use arsenic trioxide in treating the patients with acute promyelocytic leukemia (APL), and the results showed that arsenic trioxide was effective on APL patients. Further study of arsenic trioxide demonstrated that arsenic trioxide was safe and effective not only on patients with leukemia, but also on patients with many other kinds of malignant cancers. Arsenic trioxide exerted its anti-cancer effects mainly by inhibiting the cell growth and angiogenesis, inducing the cell partial cytodifferentiation and apoptosis, etc. In addition, this review looked forward to the prospect of application of gene chip, a newly developed and powerful technology in analysis of gene expression profile, in elucidation of the mechanism of arsenic trioxide on tumor.

Antineoplastic Agents↗