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dsRNA is synthesized and purified using well established protocols.
Tensions are high at the Intergalactic Space Lab because the supply of cDNA is alarmingly low. The crew of the S.S. PrimeScript space vessel receives a distress signal - can they use their powers of reverse transcription to save the day? Watch and find out!
Innate immunity mechanisms are the first line ofdefense against invading viruses. The AID/APOBEC family of cytidinedeaminases is significant in the regulation of tissue development,responding to metabolic regulation and facilitating innate immunityby restricting numerous types of virus, including HBV. Unresolvedissues are the mechanisms of AID/APOBEC-dependent specificrecognition of HBV DNA, degradation of cccDNA, and the security andavailability of experimental models, which are required for furtherinvestigation. Recent analyses of the mutations have indicated thatAID/APOBEC cytidine deaminases are significant factors in themutagenesis of human cancer genomes. A3B, which is only localizedto the nucleus, is proposed to be responsible for a largeproportion of dispersed and clustered mutations in multipledistinct cancers, including HCC. It is essential to elucidate themutational processes underlying the development of cancer, and itspotential implications on cancer etiology, prevention andtherapy.
Reverse Transcription | Thermo Fisher Scientific
NEB offers several reagents for cDNA synthesis upstream of applications such as qPCR and qRT-PCR. For your convenience, reagents are available as kits or standalone products to suit your needs.
Many RTs are available from commercial suppliers. and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. lacks 3´ → 5´ exonuclease activity. is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
Reverse Transcription (cDNA Synthesis) | NEB
The EvoScript Universal cDNA Master provides a convenient solution for time-saving cDNA Synthesis for use in two-step, real-time RT-qPCR. All reagents needed for cDNA synthesis, including primers, nucleotides, buffers, and enzymes are supplied in only two vials, minimizing pipetting efforts. The enzyme blend included in this kit has a broad temperature range and is suitable for high temperature reverse transcription. The amount of random primer and oligo(dT)18 primer included in the EvoScript Universal cDNA Master Reaction Buffer enables high cDNA yields from all regions of the RNA template.
When synthesizing a protein, DNA is transcribed into mRNA which is then translated into a protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes often contain introns which are not coding sequences, in contrast with exons, which are DNA coding sequences. During transcription, intronic RNA is excised from the RNA primary transcript and the remaining pieces of the RNA primary transcript are spliced back together resulting in processed mRNA. The mRNA code is then translated into an amino acid chain that comprises the newly made protein.
Using reverse transcriptase polymerases, DNA can be synthesised from mRNA and total RNA. Thus it is a 'complementary' copy of the mRNA, and is thus called complementary DNA (cDNA). cDNA forms the substrate for the majority of qPCR gene expression experiments.
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Reverse transcriptase - Wikipedia
Nassal M: The arginine-rich domain of thehepatitis B virus core protein is required for pregenomeencapsidation and productive viral positive-strand DNA synthesisbut not for virus assembly. J Virol. 66:4107–4116. 1992.
iScript cDNA Synthesis Kit | Life Science Research | Bio-Rad
Nguyen DH, Gummuluru S and Hu J:Deamination-independent inhibition of hepatitis B virus reversetranscription by APOBEC3G. J Virol. 81:4465–4472. 2007. : :
RNA/Reverse Transcription (RT) & cDNA Synthesis …
Research on HBV reveals that the antiviral activityof A3G requires incorporation into assembling viral particles toinhibit reverse transcription. Factors required for incorporationof the antiviral deaminase protein, A3G into HBV nucleocapsidscontinues to be investigated. It has been demonstrated that A3G andA3C bind to the HBV core protein in immunoprecipitation experiments(,). Such binding is essential forreverse transcription following infection. The binding of A3G tothe HBV core protein was only indirectly demonstrated withcoexpression of RT and pgRNA, however, not with core protein alone(,). The results are consistent with thefindings of Nguyen () that A3G was specificallyincorporated into replication-competent HBV nucleocapsids byinteracting with viral RT and RNA packaging signals. However, byfluorescence resonance energy transfer (FRET) and acceptorphotobleaching experiments, Zhao () revealed that A3G directly binds tocore proteins. Similarly, direct interaction of HBV core proteinand A3A was confirmed by proximity ligation assay and FRETanalysis. Deletion analysis was used to confirm that the centralregion of the HBV core protein (between aa 77 and 149) was involvedin the interaction with A3A ().Additionally, the A3B, A3C and A3F enzymes were also found to beassociated with the HBV capsid by interaction with the core protein(). Similar to A3G, AID wasco-immunoprecipitated with the nucleocapsid core protein. Theassumption was made that AID formed a ribonucleopro-tein complexwith the HBV core proteins and RNA during nucleocapsid assembly inwhich AID deaminated cytosines of the viral RNA, including pgRNAand ssDNA ().
CDNA Synthesis Kits | Life Science Research | Bio-Rad
The rcDNA genome is converted intocccDNA by cellular repair factors. Then, the cccDNA is transcribedto the pgRNA and subgenomic mRNAs (not shown). The mRNAs aretransported to the cytoplasm. The pgRNA is translated in thecytosol to form HBV core protein and the viral polymerase. Thesethree components assemble to form the core particle. The first(minus) DNA strand forms within the core particles via reversetranscription of the pgRNA to DNA; the pgRNA is degraded by viralRNase H as the plus strand is synthesized. (A) A3B inhibits thebinding of HnRnp K to the Enh II of HBV; (B) A3G may inhibit pgRNApackaging; (C) A3G renders HBV core protein-associated full-lengthpgRNA nuclease-sensitive; (D) A3G blocks DNA strand elongation andtargets a DNA-RNA hybrid. rcDNA, relaxed circular DNA; cccDNA,covalently closed circular DNA; pgRNA, pregenomic RNA; HBV,hepatitis B virus; APOBEC, apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like; HnRnp K, heterogeneous nuclearribonucleoprotein K; Enh II, enhancer II.
Synthesizing cDNA with Reverse Transcriptase - YouTube
A T7 promoter in reverse orientation between the polylinker and the SV40 polyA site for probe synthesis, as well as a second polylinker after the SV40 polyA site provide several sites to linearize the vector for Sp6 RNA transcription.
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