rtc isolation and purification of dna from rtcs and cell lysates
Báo cáo y học: " Revisiting HIV-1 uncoating" doc
... literature is divided as to the nature and location in the cell of HIV-1 RTCs and PICs, and most importantly as to the presence or lack of capsid in these complexes RTCs are simply defined as HIV-1 ... involves firstly the formation of the minus strand strong-stop DNA, a strand transfer event, and the synthesis of the minus strand DNA with concomitant degradation of the RNA template In the HIV-1 ... PPT (cPPT) and 3’ PPT, resist degradation by RNase H and serve as primers for synthesis of plus-strand DNA [49,53,54] Reverse transcription proceeds with synthesis of plus-strand DNA, involves...
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Nghiên cứu xây dựng quy trình phát hiện nhanh virus cúm gia cầm a h5n1 trong mẫu bệnh phẩm bằng kỹ thuật multiplex reverse transcription polymerase chain reaction
... “Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture” Virus Res, 79(1-2): 177-185 Beare AS and Webster RG ... Kida, H and Webster, RG (1996), “Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity ... “Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control”, Proc Natl Acad Sci USA, 103, pp 2845- 50 15 Chen, H., G Deng, Z Li, G Tian, Y Li, and P Jiao...
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Báo cáo khoa học: Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription ppt
... which one strand is entirely DNA and the other strand is RNA at the 5¢-end followed by a stretch of DNA, the HIV-1 and M-MLV retroviral RNases H strongly prefer to cleave the RNA strand one nucleotide ... SW, Goff S & Baltimore D (1979) A detailed model of reverse transcription and tests of crucial aspects Cell 18, 93–100 Telesnitsky A & Goff SP (1997) Reverse transcriptase and the generation of ... retroviral RNases H DNA ⁄ RNA hybrids are drawn with RNA strands in red and DNA strands in black In the internal cleavage mode, the arrows mark the sites of cleavage along the length of the hybrid...
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Báo cáo sinh học: " Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR" pdf
... real-time PCR and RTPCR offers significant advantages over standard PCR and RT-PCR for detection of DNA or RNA in terms of reduced sample handling, the time required for analysis and analytical ... carry-over amplicon DNA contamination in a sample Results Effect of UNG concentration on DNA degradation and RTPCR amplification of RNA To assess the effect of UNG on DNA degradation and RNA detection, ... Simultaneous detection of viral RNA and contaminating DNA Simultaneous detection of viral RNA and contaminating DNA Serial ten-fold dilutions of viral RNA with approximately 1,000 copies of heterologous...
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Báo cáo hóa học: " A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus" pdf
... of 1,101 fulllength NA sequences of the subtypes, of a range of host species and from different geographical locations were retrieved and aligned using Biological Sequence Alignment editor software ... subtypes obtained from a range of host species and from different geographic locations Archived clinical specimens, mainly from Queensland, Australia were also tested Efficacy of our method was ... dilutions of in-vitro transcribed RNA a band of approximately 253 bp was clearly visible with 40 femtogram of starting RNA for N1 and N7 subtypes using 36 cycles For cultured virus a band of the...
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báo cáo hóa học:" Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR" ppt
... real-time PCR and RTPCR offers significant advantages over standard PCR and RT-PCR for detection of DNA or RNA in terms of reduced sample handling, the time required for analysis and analytical ... carry-over amplicon DNA contamination in a sample Results Effect of UNG concentration on DNA degradation and RTPCR amplification of RNA To assess the effect of UNG on DNA degradation and RNA detection, ... Simultaneous detection of viral RNA and contaminating DNA Simultaneous detection of viral RNA and contaminating DNA Serial ten-fold dilutions of viral RNA with approximately 1,000 copies of heterologous...
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Báo cáo hóa học: "Rapid detection of sacbrood virus (SBV) by one-step reverse transcription loop-mediated isothermal amplification assay" pptx
... stearothermophilus (Bst) DNA polymerase (New England Biolabs Inc., Ipswich, MA), × the supplied Bst DNA polymerase buffer, 0.125 units of AMV reverse transcriptase, and µl of template RNA The RT-LAMP ... 63°C, 64°C and 65°C The DNA products from all reactions except that done at 59°C showed an obvious ladder-like pattern on the gel The intensities of the DNA products at 63°C, 64°C and 65°C were ... of SBV Figure Determination of the optimal temperature of the LAMP Determination of the optimal temperature Lane M, DL-2000 DNA marker; Lanes 1–7: LAMP carried out at 59, 60, 61, 62, 63, 64 and...
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Báo cáo khoa học: "TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus" pot
... for and 15 µl (10-100 ng/µl) were added to RT-mix for synthesis of cDNA The RT-mix consisted of µl of 5X Universal buffer, µl of 0.1 µl M DDT, µl of 10 mM dNTP, µl of reverse primer (20 pM), of ... strain were used as standard virus for detection of JEV by real-time RT-PCR The KV1899 and Anyang 300 strains were cultured in TF104 cells (a cell line cloned from MA104 cells), and the Nakayama strain ... µl, containing 25 µl of 2X Universal master mix, µl of forward primer (20 pM), µl of reverse primer (20 pM), µl of DEPC water and 15 µl of cDNA After incubation at 95oC, the cDNA was amplified by...
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Báo cáo khoa học: "Development of a Lightcycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus" pps
... demrofrep erew noitceted tcudorp dna noitacifilpmA elcyc hcae rof ces/C 02 fo pmar eht htiw nim rof C 06 ta pets noisnetxe/gnilaenna dna ces rof C 49 ta pets noitarutaned fo selcyc 55 yb dewollof ... )sgip dna swoc( slamina morf detcelloc erew IPD dna IPD neewteb detcelloc selpmas gnaborP ehT deraperp erew 2002 ni kaerbtuo DMF eht gnirud smraf gip 41 morf detcelloc seussit lailehtipe dna sdiulf ... noissucsiD IPD dna IPD neewteb RCP-TR lanoitnevnoc yb detceted eb dluoc ANR lariv dna suriv evil htob ,revewoH IPD.3 dna IPD neewteb ANR lariv rof evitisop erew sgip dna ,IPD dna IPD neewteb...
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Báo cáo y học: "Investigation of infectious agents associated with arthritis by reverse transcription PCR of bacterial rRNA" ppsx
... history, serology and culture, as appropriate (Table 1) Isolation of RNA from synovial fluid and reverse transcriptase PCR amplification of bacterial rDNA Total RNA was isolated from 200 µl untreated ... extracted from the SF samples and approximately half was then treated with DNase I, and then aliquots of both were subjected to RT-PCR using universal primers R1 and R2 for detection of bacterial ... 640, IgG 1: 640, IgA 1:160) and T -cell responses + Clinical details Universal (DNase and non-DNase) indicates the number of clones sequenced giving good quality sequence from PCR products obtained...
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Báo cáo khoa hoc:" Quantitative competitive reverse transcription polymerase chain reaction is not a useful method for quantification of CD4 and CD8 cell status during HIV infection" pptx
... were chosen that span introns of CD4 and CD8 genes in order to avoid amplification of cellular DNA contaminating RNA preparations Products are generated from cDNA and deletional mutants are constructed ... antilog of x when y = will give the concentration of unknown amount of DNA – that of CD4, CD8 or GAPDH The success of the RNA extraction and reverse transcription is determined by quantification of ... between CD4 and CD8 mRNA levels and numbers of cells expressing these proteins on their surface The formation of CD4gp160 complexes in intracellular compartments may contribute to cell surface...
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Báo cáo y học: " Variability in a dominant block to SIV early reverse transcription in rhesus monkey cells predicts in vivo viral replication and time to death" pot
... The ability of a cell to become infected by HIV/SIV requires cellular expression of diverse proteins and HIV/SIV replication can be repressed by the cellular expression of a number of restriction ... between copy number /cell of each viral DNA species and the SIVsmE660 permissivity of that cell A significant positive correlation was observed between copy number of early RT, late RT, and integrated ... state of CD4+ T cells, is not entry dependent, does not reflect the susceptibility of a subpopulation of cells, is not dependent on multiple rounds of infection, is not specific for SIVsmE660 and...
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Báo cáo y học: " A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus" pptx
... repeatability of the test 30 samples in Heilongjiang and Jilin provinces, and Fifty-one field samples from dogs, raccoons, foxes, and minks in Shandong provinces were assayed by RT-nPCR; the background of ... products were cloned and sequenced, and the sequences were aligned with the H genes of a number of CDV strains available in GenBank using the MegAlign function of the DNAStar software package Thirty ... from CDV wild-type strain and the vaccine strain, respectively Two bands of 247 and 177 bp were detected simultaneously from the mixed genomic RNA of the CDV wild-type and vaccine strains Amplification...
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Retrovirology Review BioMed Central Open Access When is it time for reverse transcription to docx
... MS RNAs ssDNA GagDNA FL DNA MS cDNAs Figure acids content of wild-type and NC mutant HIV-1 virions Nucleic Nucleic acids content of wild-type and NC mutant HIV-1 virions WT, ΔZF1, ΔZF2 and ΔZF1ZF2 ... fingers of NC contribute to the maintenance of the complete viral DNA and its IN-mediated integration into the host genome during the course of cell infection [45,79,80] Future prospects One outstanding ... promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA J Virol 1997, 71(7):5178-88 Guo J, Wu T, Anderson J, Kane...
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Báo cáo y học: " The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro" doc
... characterization of GSTand NusA-HuR (A) Analysis of the expression and purification of GSTHuR by SDS PAGE Lanes and illustrate total protein and total soluble protein after E coli cell lysis, respectively, ... solution and the molecular masses of NusA-HuR and NusARRM were determined as 82.2 and 68.9 kDa, respectively These values are within ± 15% of the predicted masses of 99 kDa for NusA-HuR and 73 ... H domain of RT and to the C-terminus of HuR (see Fig 1) Importantly, siRNA knockdown of HuR expression in HeLa P4.2 cells was reported to greatly impair both the early and late steps of viral...
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Báo cáo y học: "Subtype-associated differences in HIV-1 reverse transcription affect the viral replication" ppt
... complex (RTC) isolation and purification of DNA from RTCs and cell lysates Approximately × 106 infected Sup-T1 cells were collected and washed twice with 40 ml cold PBS For Page 14 of 18 quantitative ... analysis of cDNA and proviral DNA, total DNA was purified using the IsoQuick DNA Isolation kit and then analyzed by real-time quantitative PCR Fractionation of cells and isolation of the RTCs was ... (A and B), and backbone HIV1084i or chimeric 1084polL(NL) viruses (C and D) RTCs were purified from cell lysates DNA was isolated from RTCs and subjected to quantitative realtime PCR Levels of...
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Báo cáo y học: " Blocking premature reverse transcription fails to rescue the HIV-1 nucleocapsid-mutant replication defect" ppsx
... cells were infected with PEG-precipitated virus for h and cells were harvested at the indicated time points DNA from infected cells was isolated using a Qiagen DNA Blood Mini-Kit, and viral and ... each with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform, and ethanol precipitated Viral DNA and gRNA were quantitated to assess the initial levels of vDNA and input genomes for the ... set of cells These results are from a representative experiment The vDNA species measured were normalized for cell equivalents using CCR5 and are indicated at the bottom of panel A Schematics of...
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Báo cáo y học: "Isolated HIV-1 core is active for reverse transcription" pptx
... Early steps of retrovirus replicative cycle Retrovirology 2004, 1:9 Goff SP: Intracellular trafficking of retroviral genomes during the early phase of infection: viral exploitation of cellular pathways ... effect of elongation on core structure and it could be that the elongation of the polymerase results in shedding of capsid as suggested by the effect of NERT on virion morphology [5] Some cellular ... making DNase I-resistant full length minus-strand DNA, albeit inefficiently, without requiring capsid release Our isolation of active cores without detergent treatment was fortuitous and reproducible;...
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Báo cáo y học: "Early and transient reverse transcription during primary deltaretroviral infection of sheep" ppt
... hundred and fifty copies of each of these cloned sequences were mixed in μg of uninfected DNA Five hundred nanograms of mixed DNA were amplified for 35 cycles using 200 μM of BLV3'S and BLV3'AS ... clusters of sequences derived from each of the sheep DNA samples For each animal, cellular clones are sorted according to their date of isolation, i.e days before, 50 after seroconversion and 240 ... copies of the BLV provirus per mcg of blood DNA At copy numbers ranging from 1200 to 2400, 62.5 to 1200, and 25 to 62.5, detection was 3/4, 2/4, and 1/4, respectively Accordingly, DNA samples from...
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Báo cáo y học: " Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import" pps
... gene expression from integrated and unintegrated viral DNA [33] After 48 hours of infection, equal amounts of cells were lysed in 50 µl of luc lysis buffer and then, 10 µl of cell lysates was used ... 106 cells were lysed and serial-diluted cell lysates were analyzed by two-step Alu-PCR and Southern blot for specific detection of integrated proviral DNA from infected cells (Upper panel) The DNA ... respectively 70%, 72% and 68% of viral DNA associated with nuclear fractions (Fig 5C (upper panel, lanes and 2; and 4; and 10) and 5D) For KK240,4AE mutant, approximately 51% of viral DNA was nucleusassociated...
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