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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.

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.

Objective: To study the preparation of oligonucleotide microarray for detecting vaccinia virus., Methods: Oligonucleotide probes were designed and synthesized according to the specific genes of vaccinia virus. Sample DNA of the virus and the negative control sample were obtained and labeled by restriction display technique, followed by hybridization to the oligonucleotide microarray and scanned by Agilent scanner., Results: Strong hybridization signals were detected from the viral DNA hybridized with the microarray, but were absent in the negative sample when positive probes were not used., Conclusion: Distinct differences in the hybridization signals between the virus sample and negative sample and between the samples obtained in different phase of infection demonstrate high specificity and sensitivity of the microarray for vaccinia virus detection.

Objective: To amplify and clone the N gene of severe acute respiratory syndrome-associated coronavirus., Method: Using primer Premier 5.0 software, two pairs of nested PCR primers were designed to amplify the N gene. After purification, the amplified products were cloned into pMD18-T vectors, and the positive clones with the inserted fragments were identified by sequence analysis., Results: The amplified products was about 1 375 bp in length, and sequence analysis demonstrated that the N gene fragments had been successfully inserted into pMD18-T vectors., Conclusion: The successful amplification and cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.

Objective: To summarize and analyze the influencing factors of both success and half-year patency rate of radial artery-cephalic vein arteriovenous fistula., Methods: A retrospective analysis was made on the clinical data of 102 patients with chronic renal failure undergoing radial artery-cephalic vein arteriovenous shunt for hemodialysis during 7 years and the influence of different factors: sex, age, primary disease, incision site, and situations of vessels on the operative success rate and the half-year patency rate of the fistula was compared., Results: No significant difference in the initial success rate of operation was found between the operations performed at the two different incision sites: at the wrist and at anatomist's snuffbox. However, the operation at the wrist was better than that at anatomist's snuffbox with regard to the half-year patency rate. Both operative success rate and half-year patency rate were not influenced by patient's sex, age, and primary disease. Anastomotic hyperplasia, condition of blood vessel and anastomotic technique were important factors influencing the operative failure and fistula occlusion., Conclusion: Correct selection of incision site is of key importance for both operative success and maintenance of half-year patency of radial artery-cephalic vein arteriovenous fistula. In addition, anastomotic hyperplasia, diameter and intimal smoothness of selected blood vessel, condition of run-off vessel, operative technique, anastomotic angle, distance between artery and vein, and patient's self-protection are also considered important influencing factors.

Objective: To explore the clinical effect of percutaneous transluminal angioplasty (PTA) and stent on the subclavian steal syndrome., Methods: The clinical results of PTA and stent in the treatment of subclavian steal syndrome in 78 patients were analysed., Results: 100% interventional procedure success rate was achieved with a mean systolic arterial pressure difference of

Objective: To construct and identify the cDNA library of E.coli mRNA with poly(A) tracts., Methods: The cDNA library of E.coli was constructed by restriction display-PCR (RD-PCR) technique, followed by sequencing and bioinformatics analyses., Results: cDNA library of E.coli mRNA with poly(A) tracts was successfully constructed, and 66 gene fragments were sequenced., Conclusion: The constructed cDNA library of E.coli mRNA with poly(A) tracts contains a low rate of repetition and is of high quality.

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.

Objective: To establish a method for purifying PCR products with cetyltrimethylammonium bromide (CTAB)., Method: Selective precipitation of the PCR product was performed using CTAB, which forms compound with DNA fragment in salt solution of appropriate concentration, but not with single strain oligonucleotide or dNTPs. The precipitation could be dissolved in 1.2 mol/L NaCl while the addition of ethanol caused the desired PCR product to precipitate so as to be recovered., Result: The primers and small-molecule dNTP could be effectively eliminated after this procedure. Although the output of the purification process reached only 80% that by current reagent kit, it reduces the cost to as low as 1/8 of that normally required by the kit., Conclusion: CTAB is applicable for purification of the PCR product.

Objective: To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli., Methods: mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters. Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors., Results: More than 100 gene fragments were cloned, 30 of which were sequenced., Conclusion: Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.

Objective: To investigate the effect of probe purity on the reutilization of gene chips., Methods: Purified and unpurified probes were respectively hybridized with the gene chip and after being scanned with Scanarry lite, a laser scanning device, the gene chip was treated with stripping solution to wash off the probes. After another round of scanning under the same condition to obtain the images, the gene chip was recycled for another hybridization., Results: Hybridization of unpurified probes resulted in high background noise and obscure positive signals, which were present even after wash with stripping solution for 4 times. Hybridization using purified probes, in contrast, presented low background noise that was completely eliminated after the gene chip was washed twice before it was used again., Conclusion: The purity of probes is of great importance to the recycling of the gene chips, and hybridization with unpurified probes renders the chips impossible for reutilization.

Objective: To study the feasibility of using MTT assay to detect cell apoptosis., Methods: K562 cells were grown in RPMI 1640 medium supplemented with 10% calf serum and 4 micromol/L arsenic trioxide. Apoptosis was induced in the cultured cells by As2O3, and the cells were detected with optical microscope, DNA gel electrophoresis and MTT staining respectively., Result: MTT staining could also accurately detect cell apoptosis, by which the apoptotic cells were easily distinguished from normal cells and dead cells., Conclusion: MTT staining is simple, convenient and practical for detecting cell apoptosis.

OBJECTIVE: To clone and analyze the HIV-1gene fragments isolated by restriction digest polymerase chain reaction (RD-PCR). METHOD: All the HIV-1 gene fragments were divided into 10 subgroups and amplified by RD-PCR. The PCR products of each subgroup were purified and cloned into the T-vectors, then identified rapidly. The plasmids were extracted from positive clones and the target gene fragments were amplified and sequenced. RESULTS: Sequences analysis showed that all the fragments amplified were HIV gene. CONCLUSION: A method for cloning and identifying multiple fragments has been improved and used for restriction fragments.

As a novel technology, DNA microarray chips are being used in gene detections. To make DNA chips capable of multiple probing, ceramic slides, instead of silicon wafer or glass slides, were used as the substrate upon which DNA microarray are deposited. Our study indicates that the ceramic DNA microarray chips can be applied to multiple hybridization and detection procedures, showing high specificity. The advantages in using ceramic substrates are discussed.