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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. The reaction mix includes dNTPs, primers, template RNA, necessary enzymes, and a buffer solution.
Nucleic acid amplification is a technique used to produce several copies of a specific segment of RNA/DNA. [3] Amplified RNA and DNA can be used for a variety of applications, such as genotyping, sequencing, and detection of bacteria or viruses. [4] There are two different types of amplification, non-isothermal and isothermal. [5]
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.
The virus's nucleic acid uses the host cell's metabolic machinery to make large amounts of viral components. [2] In DNA viruses, the DNA transcribes itself into messenger RNA (mRNA) molecules that are then used to direct the cell's ribosomes. One of the first polypeptides to be translated destroys the host's DNA.
All known RNA viruses, that is viruses that use a homologous RNA-dependent polymerase for replication, are categorized by the International Committee on Taxonomy of Viruses (ICTV) into the realm Riboviria. [3] This includes RNA viruses belonging to Group III, Group IV or Group V of the Baltimore classification system as well as Group VI.
Complementarity is also utilized in DNA transcription, which generates an RNA strand from a DNA template. [4] In addition, human immunodeficiency virus, a single-stranded RNA virus, encodes an RNA-dependent DNA polymerase (reverse transcriptase) that uses complementarity to catalyze genome
Other viruses use an RNA-dependent RNAP (an RNAP that employs RNA as a template instead of DNA). This occurs in negative strand RNA viruses and dsRNA viruses, both of which exist for a portion of their life cycle as double-stranded RNA. However, some positive strand RNA viruses, such as poliovirus, also contain RNA-dependent RNAP. [49]
TMA produces RNA amplicon rather than DNA amplicon. Since RNA is more labile in a laboratory environment, this reduces the possibility of carry-over contamination. TMA produces 100–1000 copies per cycle (PCR and LCR exponentially doubles each cycle). This results in a 10 billion fold increase of DNA (or RNA) copies within about 15–30 minutes.