pcr gene sequencing and analysis

... presented in this paper expands upon those earlier studies to pro- Methods Cloning of N and NSs genes PCR was used to amplify the open reading frame of N and NSs from the pCAGGS N and NSs vectors respectively ... Figure Expression and purification of the RVFV N and NSs antigens Expression and purification of the RVFV N and NSs antigens Protein samples were mixed with reducing sample buffer and run on SDS ... Results and Discussion Cloning, expression and purification of RVFV N and NSs ELISAs have been used in the past in the diagnosis of RVFV infection in both humans and livestock [27-31], and these...

Ngày tải lên: 12/08/2014, 04:21

... using various substrates and compared with those of hPDI The recombinant cPDI and hPDI shared similar reductase, oxidase and isomerase activities PDI exhibits both chaperone and antichaperone activities ... Cl, pH 7.5, mM EDTA) The filled and open circles denote native and linear tx3a, respectively (B) Refolding carried out in buffer B (buffer A plus mM GSH and mM GSSG) and in buffer C (buffer B plus ... pGEM-T easy vector, and sequenced The nested PCR primers for 5¢-RACE amplification were based on the 3¢-end sequence, and the 5¢-end fragment was amplified using the nested primer and the 5¢-RACE adapter...

Ngày tải lên: 07/03/2014, 05:20

... hVb2.1hCb2 and hVb1hCb2 (genes kindly provided by U Utz and R P Sekaly, University of Montreal, Canada), were cloned between the NdeI and EcoRI restriction sites of the pET17b expression vector and ... equilibrated with 50 mM Tris, pH 8.5, and developed with a linear NaCl gradient Streptococcus pyogenes superantigen (SSA) The ssa-1 gene was PCR amplified from Streptococcus pyogenes DNA (ATCC 51500 strain) ... SMEZ1 and 2), three have one Cys (SEI, SEK and SPEC), two have three Cys (SEG and SPEA) and only SSA has more than that, five Cys As discussed later, the fact that SSA has two Cys residues (Cys26 and...

Ngày tải lên: 07/03/2014, 16:20

... amplified by RT -PCR with the latex poly(A)+ RNA as the template and then sequenced Finally, two cDNAs were obtained and designated HRT1 and HRT2, respectively DNA sequencing analysis Materials and methods ... 45 °C and at 72 °C with a preheat and a 10 final extension at 72 °C The resulting band of PCR products were extracted from agarose gel and subcloned into pT7Blue T vector (Novagen) for sequencing ... LI-COR Inc., Lincoln, NE, USA) Computer analysis and comparison of DNA sequences were carried out using GENETYX genetic information processing software (Genetyx Corp., Tokyo, Japan) Ó FEBS 2003...

Ngày tải lên: 07/03/2014, 21:20

... zebrafish SULT1 ST1 and ST2 mRNAs and (B) Western blot analysis of zebrafish SULT1 ST1 protein (A) Detection of zebrafish SULT1 ST1 and ST2 mRNAs in cultured zebrafish cells (lanes and 3) and whole zebrafish ... sample (lane 3) and the whole zebrafish sample (lane 4) The authenticity of the PCR products corresponding to SULT1 ST1 and cDNAs was confirmed by nested PCR using the primary PCR products as templates ... applied onto a 1.2% agarose gel and separated by electrophoresis The discrete PCR product band, visualized by ethidium bromide staining, was excised from the gel and the DNA fragment therein was...

Ngày tải lên: 08/03/2014, 02:20

... determining the optimum pH and temperature, substrate specificities, kinetic parameters and the active-site residues Results DNA and protein sequence analysis The aatB3 gene and its regulatory element ... subtilis and B circulans should belong to subgroup Ib Expression and purification of AATB3 and its mutants To produce recombinant AATB3 and the three mutant proteins, the aatB3 gene and its mutants ... AAT and PLP, the Asp232 and Lys270 residues (corresponding to Asp222 and Lys258 in E coli AAT) were replaced with Asn and His using site-directed mutagenesis to obtain the mutants D232N and K270H,...

Ngày tải lên: 14/03/2014, 23:20

... the cloning and expression of the M tuberculosis tps gene in E coli, and the production of active TPS in these cells The properties of the recombinant enzyme have been determined, and are compared ... °C The resulting PCR product was digested with NdeI and BamI, and ligated with the expression vector pET15b (Novagen), which had been linearized with the same two enzymes This generated the recombinant ... cultured with and without IPTG, and expression of a (His)6-tagged protein of the predicted size (58 kDa) was demonstrated by Western blotting (data not shown) Sequence analysis of the TPS gene (otsA)...

Ngày tải lên: 17/03/2014, 10:20

... Trypticase soy broth was from Becton Dickenson, and LB broth was from Fisher Scientific Co Sephacryl S-300 and Sephacryl S-200, and [14C]maltose and [3H]glucose, were from Amersham Pharmacia Biotech ... sugar solution and all run at the same time in the same solvent Standards of trehalose and maltose are applied to the sides of the paper to determine the locations of these sugars, and those areas ... peptides allowed the gene for TreS to be identified in the genome and its cloning and expression in E coli properties as the purified 68-kDa protein from M smegmatis On the other hand, active TreS,...

Ngày tải lên: 23/03/2014, 13:20

... sole carbon source and it is thought to be an important organism in the degradation of biomass Six T fusca cellulase genes and a xylanase gene have been cloned, expressed and characterized [8–11] ... xyloglucanase and investigate its properties and its possible role in the degradation of plant biomass Materials and methods Protein production and purification Clone KPR17269, containing the gene for ... and then heated for h at 95 °C, as described by Chirco & Brown [19] and Jung et al [12] Glucose and xylose oligomer standards were obtained from Seikagaku America or Sigma a filter with dH2O and...

Ngày tải lên: 23/03/2014, 21:20

... lane 0, protein standards (C) Protein bands after affinity chromatography and renaturing process Lanes and 2, protein bands after separation by affinity column; lanes and 4, protein bands after renaturing ... Second-strand cDNA was amplified by 5¢ PCR primer 5¢-AAGCAGTGGTATCAACGCAGAGT-3¢ and CDS III ⁄ 3¢ PCR primer, using Advantage DNA Polymerase from Clontech Screening of cathelicidin-encoding cDNAs and ... for Cc-CATH1, and 3, respectively) are shown in Figs and Alignment of three CcCATHs revealed that they share high sequence similarity with each other (Fig 1) and that Cc-CATH1 and are more closely...

Ngày tải lên: 28/03/2014, 23:20

... DNA sequencing Gene 28, 351– 359 25 Bibb, M.J., Findlay, P.R & Johnson, M.W (1984) The relationship between base composition and codon usage in bacterial genes and its use in the simple and reliable ... DNA sequencing analysis DNA sequencing analysis showed that the 1.5-kb DNA fragment contains one complete ORF with 1092 nucleotides The overall G + C content is 74%, which is typical for genes ... for min, and 72 °C for min) and an additional extension step at 72 °C for 10 The PCR product (about 1.1 kb) was purified by agarose gel electrophoresis and then subcloned into the NdeI and HindIII...

Ngày tải lên: 31/03/2014, 08:20

... cells using Western blotting, and analyzed the DPV gE gene transcription in DPV-infected cells using the real time PCR and RT -PCR Results Cloning of DPV gE gene and the correct recombinant plasmid ... the DPV gE gene using RT -PCR and real-time quantitative PCR DPV gE earliest transcripts were detected at h post-infection by RT -PCR, and markedly increased at 36 h post-infection The analysis of ... (Fig 6A) The transcription of the DPV gE gene was analyzed by real-time quantitative PCR with SYBR Green I and reverse transcription -PCR (RT -PCR) , the PCR samples amplified were detected by 1.0%...

Ngày tải lên: 12/08/2014, 04:20

... Chinese and Western isolates All these findings were based on the phylogenetic analysis of a few housekeeping genes, virulence factors genes (cagA and vacA alleles) Apart from the housekeeping genes ... (or Ks/Ka > 1), gene is under negative selection and conserved; if Ka/Ks > (or Ks/Ka < 1), gene is under positive selection and diverged (Hurst, 2002) Calculation of Ks and Ka of genes has been ... methods, various PCR based methods were used to effectively and efficiently differentiating H pylori strains Such methods include PCR based randomly amplified polymorphic DNA (RAPD) and restriction...

Ngày tải lên: 12/09/2015, 10:19

... competent cells and transformation 50 2.5.1.8 Selection and DNA sequencing 51 2.5.2 Other subclonings 52 2.5.2.1 Generation of pJG4-5-OSH6cc construct 52 2.5.2.2 Generation of YEplac-OSH7-GFP and YCplac-OSH7-GFP ... AAAAACTAAAGAA-3’ and YHRF3: 5’-CCGCTCGAGCTAATTCTTTTGGATT CCATG-3’ 2.5.1.2 PCR amplification and purification PCR was performed using Promega PCR system 39.5 µl of ddH2O, µl of 10x PCR buffer, µl ... Pharmacial Biotech, USA) The PCR products and plasmid were first separated on 1% agarose gel and the DNA bands whose size corresponding to the digested plasmid and PCR products were cut from the...

Ngày tải lên: 03/10/2015, 20:57

... further understanding of c-gliadin gene family structure Theor Appl Genet 103, 323–330 16 Hsia CC & Anderson OD (2001) Isolation and characterization of wheat x-gliadin genes Theor Appl Genet 103, ... and sequencing of PCR products The PCR product was analysed by electrophoresis through 1% agarose gel and purified using MinEluteTM Gel Extraction Kit (Qiagen, Valencia, CA, USA) The purified PCR ... c-gliadin genes and the general primary structure of c-gliadins Plant Sci 57, 141–150 15 Anderson OD & Hsia CC (2001) The wheat c-gliadin genes: characterization of ten new sequences and further...

Ngày tải lên: 20/02/2014, 01:20

... human and pufferfish (torafugu and spotted green pufferfish) IL-10 sequences Pufferfish and carp IL-10 genes, clustered together and distant from IL-20 and IL24, as recently determined from analysis ... and 57 °C (b-actin), and at 72 °C PCR products were electrophoresed on a 2.0% (w/v) agarose gel to enable detection of the specific bands Results Cloning of the IL-10 gene from carp By using PCR ... IL-10 and b-actin genes were amplified using a range (21–30) of PCR cycles Following this procedure, an optimal number of PCR cycles (24 for IL-10 and 21 for bactin) was determined and subsequently...

Ngày tải lên: 20/02/2014, 02:21

... LdEcR-R2 primers (52 °C) and with LdEcR-F3 and LdEcR-R3 primers (46 °C), respectively The first PCR with LdUSP-F1 and LdUSP-R1 primers and the second nested PCR with LdUSP-F2 and LdUSP-R1 primers ... cDNA cloning of LdEcR and LdUSP A 379-bp fragment was amplified by RT -PCR using degenerate primers, and its sequence was determined A deduced amino acid sequence of the PCR product was homologous ... The first PCR for EcR was conducted using LdEcR-F1 and LdEcR-R1 primers (annealing temperature: 48 °C) Subsequently, the second and the third nested PCR were performed with LdEcR-F2 and LdEcR-R2...

Ngày tải lên: 07/03/2014, 21:20

... most strongly in placenta and brain and to a lesser extent in heart and lung, but no signal was detected from liver, skeletal muscle, kidney and pancreas HEL cells, and Ó FEBS 2002 to a lesser ... Discussion) Fig Puri®cation and properties of wild-type and mutant Rab31 and Rab32 expressed as GST±fusion proteins Samples of platelet particulate fraction protein and of puri®ed GST and GST±Rab proteins ... platelet protein and of GST-Rab5A were included and bound [a-32P]GTP (Fig 7B) We conclude that GST±Rab31 and Fig Kinetics of GTP[S] binding by GST±Rab31 and GST±Rab32 at low and high Mg2+free...

Ngày tải lên: 08/03/2014, 16:20

... All PCR products were sizefractionated by agarose gel electrophoresis and the bands eluted, purified and subcloned in the PCRIITM TOPO vector containing the lac promoter and the b-galactosidase gene, ... tryptase (BLT and BLCT) genes, which were amplified with the proper primer pairs and subjected to restriction analysis with NspI (see Materials and methods) The two promoter sequences and 5¢-UTRs ... II and III, 60 °C for intron IV and 62 °C for intron V The PCR products were size-fractionated by electrophoresis through a 1% (w/v) agarose gel, eluted, cloned in the PCR IITM TOPO vectors and...

Ngày tải lên: 17/03/2014, 09:20

... SULTs (A) RT -PCR analysis of the expression of SULT1 ST5 and previously identified zebrafish SULT1 STs at different stages during embryogenesis and larval development onto maturity Final PCR mixtures ... zebrafish SULT displays 48 and 46% identity to dog and human SULT1B1, and lower percentage identity to other known SULTs It is generally accepted that members of the same SULT gene family share at ... SULT1 gene family [11,12], one belongs to the SULT2 gene family [13], and one appears to be independent of all known SULT gene families [14] We report here the molecular cloning, expression and...

Ngày tải lên: 23/03/2014, 15:20