extraction reverse transcriptase pcr and cycle sequencing

... phenol/ chloroform, precipitated, and reverse-transcribed using MLV RT and random primers (Invitrogen). Precipitated mRNA was detected by qPCR, using the protocol and primers described by Lal et ... program EVA/MRC (contract QLKZ-CT- 1999-00609) and the UK Medical Research Council, and were kindly provided by Dr D. Helland and Dr A.M. Szil- vay (anti-RT) and Dr G Reid (anti-p24). Mouse mono- clonal ... HIV-1 p66 reverse transcriptaseFigure 1 Identification of HuR as a partner of HIV-1 p66 reverse transcriptase. A. A yeast two-hybrid screen was carried out with HIV-1 RT-p66 as the bait, and a CEMC7...

Ngày tải lên: 13/08/2014, 05:20

... Corresponding author Abstract Background: HIV-1 reverse transcriptase (RT) is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome ... type 1 (HIV) reverse transcriptase (RT) enzyme catalyses reverse transcription of the viral RNA genome into double-stranded DNA in infected cells, a crucial early step in the virus life-cycle. RT ... synthesis to continue [14]. Plus strand DNA synthesis is initiated and following a second strand trans- fer, double-stranded viral DNA is completed. The kinetics of HIV reverse transcription during...

Ngày tải lên: 13/08/2014, 05:21

... of WT (P ≤ 0.01) and Figure of WT and mutant RTs dNTP concentration dependence of single-cycle processivity dNTP concentration dependence of single-cycle processivity of WT and mutant RTs The ... resistance to human immunodeficiency virus type with reverse transcriptase mutations K65R and K65R+M184V and their effects on enzyme function and viral replication capacity Antimicrob Agents Chemother ... specific activity and TFV susceptibility of recombinant subtype C and B HIV-1 RTs Purification, determination of specific activity and TFV susceptibility of recombinant subtype C and B HIV-1 RTs...

Ngày tải lên: 13/08/2014, 05:21

... pattern E44D /A and/ or V118I in the reverse transcriptase (RT) gene of HIV-1. .. immunodeficiency virus type 1 reverse transcriptase interacts with mutations at positions 74 and 75 via ... coefficients (phi) and Odds Ratios (OR) were calculated for 57 amino acid substitutions at 34 reverse transcriptase codons to study the association of E40F and K43E with each other and with known ... Diallo K, Marchand B, McCormick S, Gotte M: Mutations E44D and V118I in the reverse transcriptase of HIV-1 play distinct mechanistic roles in dual resistance to AZT and 3TC J Biol...

Ngày tải lên: 13/08/2014, 06:20

... Retrovirology Open Access Research Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy ... reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251) mutants with a K65R mutation in reverse transcriptase (RT), and the role of CD8+ cell-mediated ... viral RNA levels and standard immune markers [22]. Because there are some important differences in the amino acid sequence of HIV-1 and SIV RT which affect susceptibilities and the mutational...

Ngày tải lên: 13/08/2014, 09:21

... vpr, tat and nef [58-62] Our findings suggest that an intact and functional reverse transcriptase open reading frame is essential for HIV-1 replication in mothers and their infants and low degree ... 35 cycles, with the last cycle allowing for seven minutes of additional polymerization After the first round of PCR, 4µl of the first-PCR product was used for nested PCR, using inner primers and ... carried out the PCR, cloning, and sequencing VS and TH performed the sequence analysis by computer programs VS and NA participated in the experimental design, data interpretation and writing of...

Ngày tải lên: 13/08/2014, 09:21

... enzymes and reaction temperatures as follows: 37 °C (MÀMuLV reverse transcriptase), 42 °C (ProtoScriptÒ II Reverse Transcriptase), and 50 °C (WarmStart RTx Reverse Transcriptase) The Input and NP ... U/ ml penicillin and 100 mg/ml streptomycin at 37 °C in 5% CO2 as described [10–12] MÀMuLV reverse transcriptase, ProtoScriptÒ II Reverse Transcriptase, WarmStart RTx Reverse Transcriptase, RNase ... further diluted and used as PCR templates The qPCR primers were designed with Primer3 Plus program (Suppl Table S2), and the qPCR analysis was carried out using the 2x SYBR Green qPCR kit (Bimake,...

Ngày tải lên: 11/05/2020, 10:31

... METHODOLOGY Open Access Development and evaluation of a nonribosomal random PCR and nextgeneration sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens Anh ... and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing We developed and evaluated a non-ribosomal random PCR (rPCR) and ... consisted of 94 °C for and followed by 40 cycles of 94 °C for 30s, 55 °C for 30s and 72 °C for and cycle of 72 °C for Next generation sequencing library preparation and sequencing (QIAgen GmbH,...

Ngày tải lên: 18/10/2022, 18:01

... data LA, acquired and analyzed RT-PCR data KS, acquired and analyzed ELISPOT data MAP, supervised pre -validation studies for tetramer, RT-PCR, and ELISPOT and assisted writing ... using AMV Reverse Transcriptase with 25 μM of RT primer for IFNγ or CD8. Samples were stored at -15°C until further analysis. Real Time RT-PCR analysis was performed using forward and reverses ... 0.2 and 0.3 μL for IFNγ and CD8, respectively. Positive control cDNA (IFNγ and CD8 plasmid, Invitro- gen) were run in duplicate at various concentrations to generate standard curves for IFNγ and...

Ngày tải lên: 18/06/2014, 15:20

... diagnostic methods based on reverse transcription-PCR (RT-PCR) and real-time RT-PCR (RRT-PCR) are currently available for HA, but they are not well developed for NA Current NA PCR tests only identify ... participated in the extraction of RNA and edited the manuscript along with EV, WC, and CB EV was also involved with virus culture, RNA extraction and RT-PCR validation WC and CB provided clinical ... for N1 and N7 subtypes using 36 cycles For cultured virus a band of the expected size was visible in the range 103 (H5N1, 43 cycles) to 104 (H3N8, 36 cycles) copies In all cases where bands were...

Ngày tải lên: 20/06/2014, 01:20

... applied for further determination by multiplex PCR and PCR for flagellar H7 antigen detection DNA preparation for Multiplex PCR, flagellar H7 PCR and RAPD analysis E coli O157:H7 strains which ... was collected and used as a template Multiplex PCR for stx1, stx2, eaeA, and hlyA, and the flagellar H7 gene amplification Multiplex PCR for the detection of stx1, stx2, eaeA, and EHEC hlyA gene ... KCl, and 20 pmol of flagellar H7 primer The reactions were carried out with a GeneAmp PCR thermocycler The PCR condition was at 94oC for min, 65oC for min, and 72oC for The final extension cycle...

Ngày tải lên: 07/08/2014, 18:21

... α gene, between nucleotides -1,318 and +49, of 554 patients with RA from 11 Portuguese rheumatology centers serving the mainland and Azores and Madeira Islands. An association was found between ... the - 238, -308, -857, and -863 positions, and systemic manifesta- tions, functional status, radiological damage, work disability, and joint surgeries and between haplotypes and radiological damage ... (-857, -308, and -238) and systemic manifestations, radiological progres- sion, work disability, and joint surgeries. A weak association was also observed between other SNPs (-863 and -238) and DAS28,...

Ngày tải lên: 09/08/2014, 10:20

... HIV reverse transcriptase inhibitors Traditionally, in vitro efficacies are based on cumulative inhibition of multiple HIV replication cycles. We deter- mined the efficacies of HIV reverse transcriptase ... potency and the persist- ence of antiviral activity of an inhibitor. We have expanded our study to include other RTIs and to further investigate the apparent discrepancy between the potency and the ... replication cycles [16-19]. To our surprise, the EC 50 s of NVP and AZT increased by 3- and 4-folds, respectively, with pre-incubation. However, the potencies of 4'-Ed4T, DDI, and LFD4C improved...

Ngày tải lên: 10/08/2014, 05:21

... efavirenz, and nev- irapine), and also by a number of NRTIs (AZT and D4T), and protease inhibitors (PIs: indinavir and saquinavir). The antiviral effect was less pronounced with 3TC, FTC, tenofovir, and ... products, Woodland, CA), 2 mM L-glutamine and 50 μg/ ml penicillin/streptomycin (GIBCO, Grand Island, NY). PBMCs were obtained from healthy donors of humans or macaques (M. nemestrina and M. fascicularis). ... disease and was eutha- Replication kinetics of RT-SHIV, SIV, and HIV-1Figure 2 Replication kinetics of RT-SHIV, SIV, and HIV-1. Human CEMx174 cells were infected with RT-SHIV, SIV mac239 and HIV-1...

Ngày tải lên: 10/08/2014, 05:21

... of unigenes and 17.5 % of sequences) and 2981 contigs (70.7 % of unigenes and 42.1 % of sequences) assembled with... method and trehalose-thermoactivated reverse transcriptase ... resultant double-stranded cDNAs were digested with BamHI and XhoI, and ligated into the BamHI and SalI sites of a Lambda-based pFLCIII -cDNA vector [21] During the first strand cDNA synthesis ... collections by Melotto et al. [11] and Ramírez et al. [12] consisted of 5,243 and 15,333 ESTs or unigenes, respectively. However, these represented EST sequencing of three and five different cDNA libraries,...

Ngày tải lên: 11/08/2014, 11:21

... YY and DQ carried out the microarray analysis and small RNA sequencing, participated in the long npcRNA identification, and draft the manuscript MX and ZH carried out ChIP analysis, NS, CX and ... identify long npcRNAs, genome-based and transcriptbased Using genomic sequences, more than 200 candidate long npcRNAs were predicted in Escherichia coli [21], and at least 20 long npcRNA genes have ... 10b), and negative correlation in expression levels was observed between sense strand and antisense strand expression patterns in both JD8 and JD8-Pm30 (Figure 10) In addition, three long npcRNAs,...

Ngày tải lên: 11/08/2014, 11:21

... HIVPRactivityweadded200nMofthePIdarunavir (DRV) to infected and uninfected cells in the presence and absence of VRX-480773. DRV treatment impaired Gag processing (Figure 3A, lanes 3, 4 and 6) and comple- tely reversed the cytotoxic ... activation of HIV1 protease and inhibition of. .. Impact of residues in the nonnucleoside reverse transcriptase inhibitor binding pocket on HIV- 1 reverse transcriptase heterodimer ... virus type 1 reverse transcriptase A target for chemotherapeutic intervention J Biol Chem 1990, 265:8986-8988 30 Restle T, Müller B, Goody RS: RNase H activity of HIV reverse transcriptases...

Ngày tải lên: 13/08/2014, 01:20

... control, the degree of overlapping and mechanisms for coordination, and the planned timing and sequence of design activities (Krishnan and Ulrich, 2001; Browning and Ramasesh, 2007) In this ... scheduling and activity sequencing, and consists of eight chapters: Chapter 1: Introduction presents the research motivation, research gaps, research scope and objectives, and finally ... Ha and Porteus, 1995; Dahan and Mendelson, 2001; Erat and Kavadias, 2008) In this thesis, the continuous and discrete testing processes are examined separately, since the models and...

Ngày tải lên: 14/09/2015, 08:27

... 11-20 [11] K Buza and L S Thieme, "Motif-based Classification of Time Series with Bayesian Networks and SVMs," in A Fink et al (eds.) Advances in Data Analysis, Data Handling and Business Intelligences, ... K.-L and Li, W and Gao, J., Eds Proceedings 2nd International Workshop on Integrating Artificial Intelligence and Data Mining (AIDM 2007) CRPIT, 84, Gold Coast, 2007, pp 99-107 [21] Q Yang and ... mining and Analytics, 2007, pp 101-109 [7] C Gruber, M Coduro, B Sick, "Signature Verification with Dynamic RBF Networks and Time Series Motifs," in Proc of 10th Int Workshop on Frontiers in Handwriting...

Ngày tải lên: 26/02/2016, 20:11

... With two enzymes and specially developed buffer, the reaction has been successfully implemented in a single tube, including the efficient, highly specific reverse transcription and PCR The results ... MINH BỘ MƠN CƠNG NGHỆ SINH HỌC KHĨA LUẬN TỐT NGHIỆP ỨNG DỤNG PHƯƠNG PHÁP REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) ĐỂ PHÁT HIỆN Laem Singh virus TRÊN TÔM SÚ NUÔI Hướng dẫn khoa học ... sớm virus tôm sú nuôi tôm sú bố mẹ ii SUMMARY Thesis: Application of reverse transcriptase polymerase chain reaction (RT-PCR) method to detect Laem Singh Virus infected black tiger shrimp (Penaeus...

Ngày tải lên: 13/06/2018, 08:41