Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.
Cell-Free System ; DNA ; metabolism ; Oligonucleotide Array Sequence Analysis ; Protein Array Analysis ; Proteins ; metabolism ; RNA ; metabolism

OBJECTIVETo investigate DNA repair in CHL cells and HeLa cells after DNA damage induced by different oxidative agents.

METHODSCHL cells and HeLa cells were exposed to various damaging agents, CHL cells: H(2)O(2) for 25 min, K(2)Cr(2)O(7) for 105 min, doxorubicin (Dox) for 75 min HeLa cells: H(2)O(2) for 25 min, K(2)Cr(2)O(7) for 105 min; then cells were continuously cultured for 0-3 h after washing. Alkaline single cell gel electrophoresis (ASCGE) assay was used to detect DNA strand breaks.

RESULT(1) DNA strand breaks were induced in CHL cells after exposure to H(2)O(2) K(2)Cr(2)O(7) or Dox, which were repaired evidently after continuous culture for 1 h(P<0.01). The damages induced by H(2)O(2) or K(2)Cr(2)O(7) were repaired completely after culture for 2-3 h. However, the demage induced by Dox was repaired incompletely. (2) DNA strand breaks were induced also in HeLa cells after exposure to H(2)O(2) or K(2)Cr(2)O(7), which were repaired evidently after continuous culture for 0.5 h(P<0.01),and completely after culture for 1 h. (3) The regression coefficient related to the rate of comet cells and repair time was statistically different (P<0.05) between CHL cells and HeLa cells.

CONCLUSIONDNA damage induced by Dox is repaired more difficult than that induced by H(2)O(2) or K(2)Cr(2)O(7). The repair initiates immediately after DNA damage in both of cells, but more rapidly in HeLa cells than in CHL cells.

DNA ; metabolism ; DNA Damage ; DNA Repair ; HeLa Cells ; Humans ; Hydrogen Peroxide ; toxicity ; Oxidation-Reduction ; Regression Analysis

OBJECTIVETo study the frequency of telomerase gene (TERC and TERT) mutation in Northern Chinese patients with acquired bone marrow failure syndromes (BMFS).

METHODSDNA extracted from blood samples of 90 patients with BMFS (including AA, MDS, and PNH) and 45 normal controls from 4 northern hospitals was collected. TERC and TERT mutation analysis was performed by PCR.

RESULTSTwo TERC mutations (n37 A-->G, and n66 G-->C) and two TERT mutations \[n1870 G-->T (E/*)\]; and \[n1780 G-->T (S/I)\] were identified in 90 BMFS patients. Among them, 3 mutations were reported for the first time. One patient with TERT mutation, however, was finally diagnosed as DKC instead of acquired AA, making the incidence of telomerase gene mutation in northern Chinese people with acquired BMFS 3.4%, similar to that of the western country people.

CONCLUSIONThe incidence of telomerase gene mutation in northern Chinese people with acquired bone marrow failure syndromes is 3.4%, similar to that of the western country people.

DNA Mutational Analysis ; Humans ; Mutation ; RNA ; genetics ; Syndrome ; Telomerase ; metabolism

OBJECTIVESTo detect the changes of DNA ploidy of spermatogenic cells in testis and epididymis.

METHODSRight epididymides and testes from 15 fertile youth donors who died of accident were collected. Samples of spermatogenic cells in different regions of epididymis (caput, corpus and cauda) and tests were collected. DNA of spermatogenic cells were detected by flow cyctometry (FCM).

RESULTSThe haploid(1n), diploid(2n) and tetraploid(4n) spermatogenic cells were existed in different regions of epididymis and testis. The 1n cells increased from (24.87 +/- 7.28)% in testis to (96.33 +/- 1.58)% in epididymis cauda, there were significant differences among regions of testis and epididymis caput, corpus(P < 0.01), and the difference among regions of epididymis corpus and epididymis cauda were also significant(P < 0.05). While the percentages of 2n and 4n cells decreased from (63.07 +/- 8.96)% and (9.43 +/- 3.83)% in tesits to (2.47 +/- 0.93)% and (1.17 +/- 0.95)% in epididymis respectively. There was significant difference of 2n cells between testis and epididymis caput, corpus(P < 0.01), and was also remarkable difference between epididymis corpus and cauda (P < 0.05). There was no difference of 4n cells between testis and epididymis caput(P > 0.05). There were significant difference among regions of epididymis caput, corpus and cauda(P < 0.05).

CONCLUSIONSThe percentage of immature spermatogenic cells decreased along with passing through the epididymis.

Adult ; DNA ; analysis ; Epididymis ; metabolism ; Flow Cytometry ; Humans ; Male ; Spermatogonia ; metabolism ; Testis ; metabolism

OBJECTIVETo study the genotoxicity effect of environmental tobacco side-stream smokes (ETSS) on oxidative DNA damage and its molecular mechanism.

METHODSDNA adduct 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. The level of 8-OHdG in DNA exposed to ETSS was detected by high performance liquid chromatography with electrochemical detection. Organic and inorganic components in ETSS were analyzed by gas chromatography-mass spectrum and atomic absorption spectrum respectively.

RESULTSParticle matters (PMs) and volatile organic compounds (VOCs) in ETSS could directly induce oxidative DNA damage and formation of 8-OHdG. There were 123 and 84 kinds of organic components in PMs and VOCs respectively, and 7 kinds of inorganic components in ETSS. Some components, especially quinones and polyphenols in ETSS, could produce free radicals in vitro by auto-oxidation without any biological activity systems, and with the catalytic reaction of metals, the DNA adduct 8-OHdG was produced.

CONCLUSIONETSS have biological oxidative effect on DNA in vitro and in vivo, and expressed direct genotoxicity. 8-OHdG is a valuable biomarker of oxidative DNA damage.

Animals ; Biomarkers ; analysis ; Cattle ; DNA ; drug effects ; metabolism ; DNA Adducts ; analysis ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; analysis ; Female ; Lung ; chemistry ; metabolism ; Metals, Heavy ; analysis ; Organic Chemicals ; analysis ; Oxidation-Reduction ; Rats ; Tobacco Smoke Pollution ; adverse effects ; analysis

OBJECTIVETo establish and improve the method of bisulfite sequencing for methylation status of imprinted genes in single human oocytes.

METHODSSingle superovulated immature human oocyte was embedded into low melting point agarose, followed by bisulfite treatment and polymerase chain reaction (PCR) amplification of the H19 and MEST genes. The PCR products were then subjected to TA cloning and sequencing to determine the methylation status.

RESULTSWith the modified methods of embedding and bisulfite treatment, we achieved a high PCR success rate of 82.46%, with the somatic cell contamination rate as low as 7.14%. The sequencing results showed no non-CpG cytosine and exact conformity to the theoretical sequences.

CONCLUSIONThe bisulfite sequencing method we used to determine the methylation status of imprinted genes at the single-cell level was highly efficient and reliable, which can serve as a foundation for the further study of the influences of human assisted reproductive technology on genomic imprinting.

DNA Methylation ; Female ; Genomic Imprinting ; genetics ; Humans ; Oocytes ; metabolism ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

OBJECTIVETo select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library.

METHODSWith a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique.

RESULTSAfter 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133.

CONCLUSIONWe have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.

AC133 Antigen ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; DNA, Single-Stranded ; Glycoproteins ; metabolism ; Humans ; Neoplastic Stem Cells ; metabolism ; Peptide Library ; Peptides ; metabolism ; Protein Binding ; Sequence Analysis, DNA

OBJECTIVETo assess the accuracy and reliability of the nest-PCR-sequence specific primer(SSP) method in HLA-A site genotyping of single blastomeres retrieved from human pre-implantation embryos.

METHODSBy nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits.

RESULTSThe amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from in vitro fertilization(IVF) surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2%(43/55) and 93.8%(61/65), respectively. And 86.7%(104/120) in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents' homozygosity, the left 63 blastomeres had accurate HLA genotyping. Among these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents(93.6%), 3 blastomeres lost one of parents' alleles(4.8%), and only one blastomere had two more than parents' alleles(1.6%).

CONCLUSIONThe above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in pre-selecting HLA-identical sibling for allogeneic cord blood cell transplantation.

Base Sequence ; Blastomeres ; metabolism ; DNA ; analysis ; DNA Fingerprinting ; methods ; DNA Mutational Analysis ; Female ; HLA Antigens ; analysis ; HLA-A Antigens ; analysis ; genetics ; Histocompatibility Antigens Class I ; analysis ; genetics ; Humans ; Male ; Polymerase Chain Reaction ; methods ; Single Person

Base Sequence ; DNA ; chemistry ; DNA-Directed DNA Polymerase ; metabolism ; Fluorescent Dyes ; chemistry ; Humans ; Molecular Sequence Data ; Nanostructures ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods