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As a result, the fusion of the viral membrane and the plasma membrane occurs. Fusion of the membranes leads to the deposition of the virion content in the cytoplasm of the cell. After entering the cytoplasm, viral RNA is copied into a single dsDNA molecule by reverse transcriptase.
Using intact, high-quality RNA and a sequence-specific primer will produce the best results. Once a one-step RT-PCR kit with a mix of reverse transcriptase, Taq DNA polymerase, and a proofreading polymerase is selected and all necessary materials and equipment are obtained a reaction mix is to be prepared.
A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription.Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes.
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (cDNA) with reverse transcriptase.
Identical LTR sequences at either end of a retrotransposon. A long terminal repeat (LTR) is a pair of identical sequences of DNA, several hundred base pairs long, which occur in eukaryotic genomes on either end of a series of genes or pseudogenes that form a retrotransposon or an endogenous retrovirus or a retroviral provirus.
The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reduced RNase H activity suited for transcription of longer RNAs. [11] The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature).
All members of Riboviria contain a gene that encodes for an RNA-dependent polymerase, also called RNA-directed polymerase. There are two types of RNA-dependent polymerases: RNA-dependent RNA polymerase (RdRp), also called RNA replicase, which synthesizes RNA from RNA, and RNA-dependent DNA polymerase (RdDp), also called reverse transcriptase (RT), which synthesizes DNA from RNA. [3]
Transcription-mediated amplification (TMA) is an isothermal (performed at constant temperature), single-tube nucleic acid amplification system utilizing two enzymes, RNA polymerase and reverse transcriptase. "Amplification" means creating many more copies of a strand of nucleic acid than was present at first, in order to readily detect it or ...
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