OBJECTIVETo determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase.

METHODSTwo-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated.

RESULTSExo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase.

CONCLUSIONThese data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers ; chemistry ; genetics ; Exonucleases ; metabolism ; Humans ; Phosphorothioate Oligonucleotides ; chemistry ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciNbeta and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.
Child ; DNA Primers/chemistry/metabolism ; DNA, Bacterial/chemistry/genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Pneumococcal Infections/microbiology ; Serotyping ; Streptococcus pneumoniae/*classification/genetics/isolation & purification

Outwardly rectifying swelling-activated chloride conductance (ICl,Swell) in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning (IP). But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones (e.g. ClC-2, ClC-3, ClC-4 and ClC-5) were determined using RT-PCR and Western blot analysis. Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4' isothiocyanato-2,2-disulfonic acid (DIDS), 5-nitro-2(3-phenylroylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of I(Cl,Swell), including outward rectification, activation due to cell volume change, sensitivity to DIDS, IAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.
Biophysics/methods ; Chlorides/*chemistry ; Chlorides/metabolism ; DNA Primers/chemistry ; Electrophysiology/methods ; Gene Expression Regulation ; Glycolates/pharmacology ; Heart Ventricles/*cytology ; Ischemic Preconditioning ; Muscle Cells/*cytology ; Osmosis ; Sequence Analysis, DNA

BACKGROUND: Human neutrophil antigens (HNAs) are involved in autoimmune and alloimmune neutropenia and transfusion-related acute lung injury. The HNA-1 system is important in immunogenetics, and allele frequencies have been described in different populations. This study investigated the frequency of FCGR3B alleles encoding HNA-1a, HNA-1b, and HNA-1c among Thai blood donors and compared these frequencies with those previously reported for other populations. METHODS: Eight hundred DNA samples obtained from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, and the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, were included. Samples were simultaneously typed for each FCGR3B allele using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. RESULTS: The frequencies of FCGR3B*1, FCGR3B*2, and FCGR3B*3 alleles in central Thai blood donors were 0.548, 0.452, and 0.004, respectively; only FCGR3B*1 and FCGR3B*2 alleles were found in northern Thai blood donors (0.68 and 0.32, respectively). Compared with other Asian populations, central Thais had higher frequencies of the FCGR3B*2 allele (P<0.001), while the frequencies of the FCGR3B*1 and FCGR3B*2 alleles in northern Thais were similar to those previously reported in Taiwanese and Japanese populations. In contrast, the frequencies of the FCGR3B*1 and FCGR3B*2 alleles in the northern Thai population were statistically different from those observed in central Thai, Korean, German, and Turkish populations. CONCLUSIONS: FCGR3B allele frequencies were significantly different between central and northern Thai blood donors. Our in-house PCR-SSP method is a simple, cost-effective, and convenient method for FCGR3B allele detection.
Asian Continental Ancestry Group/*genetics ; *Blood Donors ; DNA/analysis ; DNA Primers/chemistry/metabolism ; GPI-Linked Proteins/genetics ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; Receptors, IgG/*genetics ; Thailand

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.
Blood Stains ; Body Fluids/chemistry* ; DNA/analysis* ; DNA Primers ; Forensic Medicine/methods* ; Gene Expression Profiling ; Humans ; RNA/analysis* ; RNA, Messenger/metabolism* ; Reverse Transcriptase Polymerase Chain Reaction/methods* ; Saliva/chemistry* ; Semen/chemistry*

OBJECTIVETo investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant.

METHODSNonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302. The pro-UK mutant and native pro-UK were transfected into dhfr(-)-CHO cells, and serum-free culture supernatant was harvested and incubated at 4 degrees C and 37 degrees C, respectively. The pro-UK activity in culture supernatant was measured by the optical density (OD) increase with time (12 hours) at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured.

RESULTSAfter 48 hours of incubation at 4 degrees C, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37 degrees C decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37 degrees C. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4 degrees C and 37 degrees C.

CONCLUSIONHigher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.

Amino Acid Substitution ; Animals ; Asparagine ; chemistry ; Base Sequence ; CHO Cells ; Cricetinae ; Cricetulus ; Culture Media ; DNA Primers ; genetics ; Enzyme Stability ; Glycosylation ; Humans ; In Vitro Techniques ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; chemistry ; genetics ; metabolism

OBJECTIVEBloom's syndrome is an autosomal recessive disorder characterized by genomic instability and a predisposition to many cancers. Mutations of the BLM gene (encoding a BLM helicase) may form a structure of the etiology of this disease. As a global pollutant, mercury poses a major threat to human health. The current study was conducted to elucidate the effects of Hg(2+) on the structure and activity of BLM642-1290 recombinant helicase, and to further explore the molecular mechanisms of mercury toxicity to the DNA helicase.

METHODSThe effects of Hg(2+) on biological activity and structure of BLM642-1290 recombinant helicase were determined by fluorescence polarized, ultraviolet spectroscopic, and free-phosphorus assay technologies, respectively.

RESULTSThe helicase activity, the DNA-binding activity, and the ATPase activity of BLM642-1290 recombinant helicase were inhibited by Hg(2+) treatment. The LMCT (ligand-to-metal charge transition) peaks of the helicase were enhanced with the increase of the Hg(2+) level. The LMCT peaks of the same concentration of helicase gradually increased over time.

CONCLUSIONThe biological activity of BLM642-1290 recombinant helicase is inhibited by Hg(2+) treatment. The conformation of the helicase is significantly altered by Hg(2+). There exist two binding sites between Hg(2+) and the helicase, which are located in the amino acid residues 1063-1066 and 940-944 of the helicase, respectively.

Adenosine Triphosphatases ; metabolism ; Base Sequence ; DNA Primers ; Fluorescence Polarization ; Humans ; Mercury ; toxicity ; Protein Conformation ; RecQ Helicases ; chemistry ; drug effects ; metabolism ; Recombinant Proteins ; chemistry ; drug effects ; metabolism ; Spectrophotometry, Ultraviolet ; Structure-Activity Relationship

Mitochondrial cytochrome oxidase III (COIII) of duck was successfully amplified by PCR-mtDNA with duck muscle DNA as the template (GenBank Accession No. DQ655706). Cloning sequence analysis shows that the 784 bp nucleotides of COIII gene were contained. Through homology analysis, we confirmed that the cytochrome oxidase III (COIII) was relatively conservative. The method of PCR-mtDNA can be designed to detect the components of duck origin. And then, the method of PCR can be applied to amplify with the muscle DNA of various animal and feedstuff as the template, repeated verification, the primer (P3, P4) with strong specificity and good stability is screened, which can only amplify the sequence of duck. The special sequence contains 226 bp, the amplified product of 226 bp was sequenced and analyzed, it showed 100% homology with duck mtDNA COIII gene, which proved the accuracy of the special primer. The test that used different concentration of DNA with P3 and P4 is the sensitive experiment by PCR. The result showed that the primer has much specialty and rather sensitivity. So it is a way to detect the duck origin in the muscle of various animal and feedstuff.
Animal Feed ; analysis ; Animals ; DNA Primers ; genetics ; metabolism ; DNA, Mitochondrial ; genetics ; metabolism ; Ducks ; genetics ; Electron Transport Complex IV ; genetics ; Mitochondria, Muscle ; genetics ; Muscle, Skeletal ; chemistry ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

To investigate the distribution of the genes of two major metallo-beta-lactamases (MBL; i.e., IMP and VIM) and class 1 integrons (intI) in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for bla ( IMP-1 ), bla ( VIM ) and bla ( VIM-2 ) genes. The MBL-positive isolates were further assessed for class 1 integrons by PCR using specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the bla ( VIM ) gene was found in 81.5% (53/65) of all isolates, bla ( VIM-2 ) gene was found in only 1 isolate and the intI gene was observed in 45.3% (24/53) of bla ( VIM )-positive isolates. One isolate carried simultaneously both bla ( IMP-1 ) and intI genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM beta-lactamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P. aeruginosa.
Anti-Bacterial Agents/pharmacology ; China ; DNA Primers/chemistry ; Drug Resistance, Bacterial ; Gene Expression Regulation, Bacterial ; Imipenem/*pharmacology ; Integrons ; Microbial Sensitivity Tests ; Models, Genetic ; Pseudomonas Infections/genetics ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/*metabolism ; Sequence Analysis, DNA ; beta-Lactamases/*metabolism

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma (BTCC). The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR (MSP). The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.
Blotting, Western ; Carcinoma, Transitional Cell/metabolism ; DNA Methylation ; DNA Primers/chemistry ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Promoter Regions, Genetic ; Tumor Suppressor Proteins/*biosynthesis ; Tumor Suppressor Proteins/*genetics ; Urinary Bladder Neoplasms/*metabolism