Beta-Thalassemia is a blood disease with beta-globin deficiency caused by a directly down-regulation in the synthesis of structurally normal beta chains. Vent DNA polymerase is a high-fidelity thermophilic DNA polymerase, and the fidelity 5 - 15 times higher than Taq DNA polymerase (1/31,000 vs. 1/290 - 1/2,400). Using ARMS PCR (Amplification Refractor Mutation System-PCR) with Vent DNA polymerase, we found that 9/28 (32%) of the tested beta-Thalassemia patients had the mutation at codon 17 and 4/28 (14%) at codon 41/42. This method can be applied for a rapid prenatal diagnosis of beta-Thalassemia disease and has public health significance.
beta-Thalassemia ; DNA ; Polymerase Chain Reaction ; Diagnosis

OBJECTIVES: Peripheral blood-buffy coat fractions (N = 14, 956) have been stored at -70degrees C in the headquarter of the Korean Multicenter Cancer Cohort (KMCC), since 1993. To study the future molecular etiology of cancers using specimens of the cohort, properly stored specimens are necessary. Therefore, the DNA-viability of the buffy coat samples was investigated. METHODS: Buffy coat fraction samples were randomly selected from various collection areas and years (N = 100). The DNA viability was evaluate from the UV-absorbent ratios at 260/280nm and the PCR for beta-globin was performed with genomic DNA isolated from the buffy coat. RESULTS: PCR products were obtained from 85 and 98% of the C and H area-samples, respectively, using 50 or 100mul of the buffy coat. There were significant differences in the yields of the PCR-amplifications from the C and H areas (p < 0.05), which was due to differences in the homogenization of the buffy coat fractions available as aliquots. The PCR-products were obtained from all of the samples (N = 7) stored at the C area-local center, but the other aliquots stored at the headquarter were not PCR-amplified. Therefore, the PCR products in almost all the samples, even including the DNA-degraded samples, were obtained. In addition, an improvement in the DNA isolation, i.e. approx. 1.6 fold, was found after using extra RBC lysis buffer. CONCLUSIONS: PCR products for beta-globin were obtained from nearly all of the samples. The regional differences in the PCR amplifications were thought to have originated from the different sample-preparation and homogenization performance. Therefore, the long term-stored buffy coat species at the KMCC can be used for future molecular studies.
beta-Globins ; Cohort Studies* ; DNA* ; Polymerase Chain Reaction

OBJECTIVE: To detect potential mutations of HEXB gene in an infant with Sandhoff disease (SD). METHODS: Genomic DNA was extracted from peripheral blood sample of the infant. All coding exons (exons 1 to 14) and splicing sites of the HEXB gene were subjected to PCR amplification and direct sequencing.PubMed Protein BLAST system was employed to analyze cross-species conservation of the mutant amino acid. PubMed BLAST CD-search was performed to identify functional domains destroyed by thecandidate mutations. Impact of the mutations was analyzed with software including PolyPhen-2, Mutation Taster and SIFT. Whole-exome sequencing was carried out to identify additional mutations. RESULTS: The infant was found to carry compound heterozygous mutations c.1652G>A(p.Cys551Tyr) and c.1389C>G (p.Tyr463*) of the HEXB gene. The c.1389C>G (p.Tyr463*) mutation may lead to destruction of two functional domains in β subunit of the Hex protein. The c.1652G>A(p.Cys551Tyr) mutation, unreported previously,was predicted to be probably damaging by Bioinformatic analysis. CONCLUSION Compound heterozygous mutations c.1652G>A(p.Cys551Tyr) and c.1389C>G (p.Tyr463*) in the HEXB gene probably underlie the disease in this patient.
DNA Mutational Analysis ; Exons ; Heterozygote ; Humans ; Infant ; Mutation ; Polymerase Chain Reaction ; Sandhoff Disease ; genetics ; beta-Hexosaminidase beta Chain ; genetics

A clinical isolate of Klebsiella pneumoniae K7746 produced the extended-spectrum beta-lactamase (ESBL) SHV-12. A 6.6 kb BamHI fragment containing the blaSHV-12 gene of K7746 strain was cloned into pCRScriptCAM vector resulting in the recombinant plasmid p7746-C1. The restriction map of 3.6 kb inserted DNA and sequences immediately surrounding blaSHV-12 of p7746-C1 were homologous to plasmid pMPA2a carrying blaSHV-2a. In addition, both blaSHV-12 and blaSHV-2a were expressed from a common hybrid promoter made of the -35 region derived from the left inverted repeat of IS26 and the -10 region from the blaSHV promoter itself. The results indicate that blaSHV-12 and blaSHV-2a may have evolved from a common ancestor in the sequential order of blaSHV-2a first, followed by blaSHV-12. Furthermore, by the PCR mapping method using primers corresponding to the IS26 and blaSHV, the association between IS26 and blaSHV was studied in 12 clinical isolates carrying blaSHV-2a, 27 clinical isolates carrying blaSHV-12, and 5 reference strains carrying blaSHV-1 to blaSHV-5. All 39 strains carrying blaSHV-2a or blaSHV-12 were positive by the PCR, providing confirmative evidence that IS26 has been involved in the evolution and dissemination of blaSHV-2a and blaSHV-12. But 5 reference strains carrying blaSHV-1 to blaSHV-5 were negative by the PCR. Therefore, we concluded that the molecular evolutionary pathway of blaSHV-2a and blaSHV-12 may be different from that of other blaSHV-ESBL, e.g., blaSHV-2, blaSHV-3, blaSHV-4, and blaSHV-5.
beta-Lactamases ; Clone Cells ; DNA ; Klebsiella pneumoniae ; Plasmids ; Polymerase Chain Reaction

OBJECTIVETo explore the effects of DNA polymerase &beta; expression level on the genotoxicity and genetic instability induced by benzo(a)pyrene (BaP),and provide experimental the basis for further study on the carcinogenic molecular mechanism of BaP.

METHODSThree kinds of cell lines with the identical genetic background, pol&beta; wild-type cells (pol&beta;+/+), pol&beta; null cells (pol&beta;-/-) and pol&beta; overexpression cells (pol&beta; oe) were applied as cellular models. The oxidative damage, genotoxicity and genetic instability induced by BaP were analyzed by using different methods respectively.

RESULTSCell viability and colony forming ability of 3 kinds of cell lines exposed to BaP decreased with BaP. After treated with 5 and 20 µmol/L concentration of BaP, fluorescence intensity of pol&beta;-/- cell line was significantly higher than that of other two cell lines (P < 0.05). When treated with 5.00 µmol/L and 20.00 µmol/L concentration of BaP, the SOD activities (76.56 ± 2.84 and 62.78 ± 4.28 U/mg pro) of pol&beta;-/- cell line were significantly lower than that (84.85 ± 3.59) of control group and those (85.21 ± 3.20 and 76.90 ± 3.38 U/mg pro) of pol&beta;+/+ cell line. In 20.00 µmol/L BaP group. SOD activity (82.59 ± 4.64 U/mg pro) of pol&beta; oe cell line was lower than that (88.58 ± 6.77 U/mg pro) of control but higher than that of pol&beta;+/+ cell line (P < 0.05). In 1.25, 5.00 and 20.00 µmol/L concentration BaP groups, the micronucleus rates of pol&beta;-/- cell line were much higher than those of pol&beta;+/+ cell line (P < 0.05). In 5.00 and 20.00 µmol/L concentration BaP groups, the micronucleus rates of pol&beta; oe cell line were significantly lower than those of pol&beta;+/+ line (P < 0.05). In 5.00 and 20.00 µmol/L concentration BaP groups, HPRT gene mutation frequencies (26.16 × 10(-6) and 37.51 × 10(-6); 27.68 × 10(-6) and 38.63 × 10(-6)) in pol&beta;-/- cells and pol&beta; oe cells were significantly higher than those (19.76 × 10(-6) and 24.78 × 10(-6)) of pol&beta;+/+ cells (P < 0.05).

CONCLUSIONPol&beta; could play a role in protecting the cells from the genotoxicity and genetic instability induced by BaP, and the normal expression level of pol&beta; was indispensable for maintaining genome stability.

Animals ; Benzo(a)pyrene ; toxicity ; Cell Line ; DNA Damage ; DNA Polymerase beta ; metabolism ; Mice ; Micronucleus Tests ; Mutation Rate

PURPOSE: To identify Bacterial translocation (BT) from the gut to the blood in the critically ill patients by using the polymerase chain reaction (PCR) to confirm the sensitivity of PCR in the detection of intestinal bacterial deoxyribonucleic acid (DNA) in human blood. Further, to determine the relationship between the identification of BT and the prognosis of these patients. METHODS: The oligonucleotide primers used to amplify bacterial DNA from whole blood were the beta-galactosidase (BG) gene of E. coli, DNA coding for 16S ribosomal RNA (16S rRNA), and the glutamine synthase gene of Bacteroides fragilis (BFR). DNA was extracted from the blood of 45 cases of critically ill patients and 10 controls. PCR techniques were used to amplify the genes from E. coli, Bacteroides fragilis, and a region of 16S ribosomal RNA found in many gram-negative and positive bacteria. RESULTS: Bacterial DNA genes were not detected in any of the controls, but were found all in 6 cases of patients with positive blood cultures. Of the 39 cases with no growth in their blood culture, 11 cases in BG and BFR, and 13 cases in 16S rRNA had positive findings in bacterial DNA PCR. Fifteen cases (33%) in BG, 19 cases (42%) in BFR, and 16 cases (35.5%) in 16S rRNA of the critically ill patients had detectable bacterial DNA in their blood. Of those with a positive PCR, MOF developed in 11 cases (57.9%) and of these, 10 subsequently died of MOF. One case (3.8%) in the negative PCR was developed and died of MOF. Patients having positive translocated bacterial DNA had a worse prognosis than the group with a negative DNA. CONCLUSION: In order to confirm BT, the PCR method for detecting bacterial DNA in the blood of critically ill patients is more sensitive than blood cultures. BT from the gut can be a major factor in the development of multiple organ failures in critically ill patients. Therefore, early detection of BT with PCR can play a major role in the treatment of critically ill patients.
Bacteria ; Bacterial Translocation* ; Bacteroides fragilis ; beta-Galactosidase ; Clinical Coding ; Critical Illness* ; DNA ; DNA Primers ; DNA, Bacterial ; Glutamine ; Humans ; Multiple Organ Failure ; Polymerase Chain Reaction ; Prognosis* ; RNA, Ribosomal, 16S

PURPOSE: Urea-splitting organisms have known to participate in the formation of infected calculi, but sometimes, causative organisms were not detected in urine culture. We compared results of polymerase chain reaction which detect urease gene in infected calculi to urine culture, to follow pathogens which involved in the formation of infected calculi and establish preventive antimicrobial therapy. MATERIALS AND METHODS: Urine culture were performed in 25 patients who were diagnosed as infected calculi. The DNA was extracted from the PBS solution that was used for washing stones and lysis solution which inserted after calculi crushing. And then, PCR were performed with universal primers for beta-globin gene, 16S ribosomal RNA gene, and primers for urease gene of Proteus mirabilis and Ureaplasma urealyticum which synthesized by order. Stone analysis was performed using infrared spectroscopy. RESULTS: Proteus mirabilis and Ureaplasma urealyticum were not detected in urine culture. All results of PCR to beta-globin gene and 16S ribosomal RNA gene were negative in calculi washing solution. In 18 of 25 cases(72.0%), the result of PCR to 16S ribosomal RNA gene were positive in calculi lysis solution. Each 2 and 3 of 18 cases(total; 27.7%) which were positive in PCR to 16S ribosomal RNA gene, Proteus mirabilis and Ureaplasma urealyticum were cultured respectively. From 16 cases which were available to perform infrared spectroscopic stone analysis, 7 cases were shown to have specfic absorbance band of infected calculi and were positive in PCR to 16S ribosomal RNA gene. CONCLUSIONS: We detected urea-splitting organisms in crushed calculi specimen using PCR. It suggests that PCR for urea-splitting organisms will be helpful to identify process of infected calculi and causative organisms.
beta-Globins ; Calculi* ; DNA ; Humans ; Polymerase Chain Reaction* ; Proteus mirabilis ; RNA, Ribosomal, 16S ; Spectrum Analysis ; Ureaplasma urealyticum ; Urease*

BACKGROUND: Of the plasmid-mediated AmpC beta-lactamases (ABLs), CMY-2 is the most prevalent and is distributed in many countries. However, little is known about the emergence and characteristics of CMY-2 among Escherichia coli isolates in Korea. The aims of this study were to detect the emergence of the CMY-2 beta-lactamase in clinical isolates of E. coli from various regions in Korea. METHODS: Eighteen cefoxitin non-susceptible isolates of 1, 130 consecutive, nonrepeat isolates of E. coli at five university hospitals were tested for antimicrobial susceptibility by the broth microdilution method. The cefoxitin non-susceptible isolates were further investigated by AmpC disk tests, double disk synergy (DDS) tests, isoelectric focusing, CMY-2-specific PCR, DNA sequencing, and plasmid analysis. RESULTS: Seven (0.6%) isolates of plasmid-mediated ABL-producing E. coli were found at three of the five hospitals; all seven isolates produced CMY-2 beta-lactamase and one of the isolates was also tested positive by the DDS test. All isolates demonstrated different plasmid patterns by plasmid analysis. CONCLUSIONS: Our data indicate that CMY-2-producing E. coli has emerged and is prevalent in the medical institution in Korea. Therefore, constant surveillance is needed to prevent its further spread.
beta-Lactamases ; Cefoxitin ; Escherichia coli* ; Hospitals, University ; Isoelectric Focusing ; Korea ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis, DNA

After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N-terminal and C-terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants' xylanase in their families and structures. It couldn't express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap-PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynIII was acquired. The cDNA was inserted into pETBlue-2 vector and transformed into E. coli DE3 pLacI cell. Xyn III could be expressed in the transformed cell under the conditions of 37 degrees C, 1 mmol/L IPTG induced for 3h. Low temperature (15 degrees C), long time(64h) induction(0.2 mmol/L IPTG) could enhance xynIII activity.
Cloning, Molecular ; DNA, Complementary ; chemistry ; Endo-1,4-beta Xylanases ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Trichoderma ; genetics

OBJECTIVETo explore the effect and mechanism of DNA polymerase &beta; expression level on cell apoptosis and mitochondrial membrane potential induced by hydroquinone.

METHODSPol&beta; wild-type cells (pol&beta;+/+), pol&beta; overexpressed cells (pol&beta; oe) and pol&beta; null cells (pol&beta;-/-) were applied as a model cell system, The effect of cell apoptosis and mitochondrial membrane potential induced by different doses of hydroquinone were analyzed by flow cytometry. The ROS and ·OH assay kit were used to examine the cellular ROS and ·OH level. The activity of cellular SOD and GSH-Px were tested by Chemiluminescence method after exposed to different concentrations of hydroquinone.

RESULTSWith the dose of hydroquinone increased, the rate of apoptosis and falling of mitochondrial membrane potential (ΔΨm) in cells were increased compared with the control. When compared with pol&beta;+/+ cells, the rate of apoptosis in pol&beta;-/- cells exposed to 20.00, 40.00, 80.00 µmol/L hydroquinone increased and the rate of apoptosis in pol&beta; oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone decreased (P < 0.05). Compared with pol&beta;+/+ cells (20.60% ± 0.57%, 37.95% ± 0.64%, 44.50% ± 1.27%, 57.55% ± 1.06%), the rate of cell which undergone mitochondrial depolarization in pol&beta;-/- cells treated with 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (33.60% ± 1.55%, 46.05% ± 1.77%, 52.75% ± 2.05%, 75.20% ± 0.56%) increased. The rate of cell which undergone mitochondrial depolarization in pol&beta; oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (16.05% ± 1.20%, 29.80% ± 1.21%, 35.15% ± 1.06%, 53.80% ± 0.85%) decreased (P < 0.05). When compared with pol&beta;+/+ cells, fluorescent intensity of pol&beta;-/- cells treated with different dosages of hydroquinone increased, while which of pol&beta; oe cells decreased (P < 0.05). Compared with pol&beta;+/+ cells, ·OH level of pol&beta;-/- cells treated with 20.00, 40.00 µmol/L hydroquinone significantly enhanced, while which of pol&beta; oe cells decreased sharply (P < 0.05). Under the same concentrations of hydroquinone, the activity of SOD and GSH-Px were decreased most rapidly in pol&beta;-/- cells. The activity of SOD and GSH-Px in pol&beta; oe cells decreased slower than in the pol&beta;-/- cells.

CONCLUSIONHydroquinone could induced apoptosis by the generation of ROS and decrease of ΔΨm; pol&beta; could protect cells from apoptosis induced by hydroquinone through decrease of ROS level and depolarization of mitochondria.

Animals ; Apoptosis ; drug effects ; Cells, Cultured ; DNA Polymerase beta ; metabolism ; Hydroquinones ; toxicity ; Membrane Potential, Mitochondrial ; drug effects ; Mice