Reverse transcriptase polymerase chain reaction

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The reverse transcriptase polymerase chain reaction ( RT-PCR ) is the combination of two methods of molecular biology - the use of reverse transcriptase (RT) and the polymerase chain reaction (PCR) - to detect RNA, e.g. B. the gene expression of specific genes in cells , tissues and blood serum or ribozymes , ribonucleoproteins or the genome of RNA viruses . RT-PCR is used in research and diagnostics .

The abbreviation RT-PCR sometimes also refers to Real Time Quantitative PCR , which can lead to confusion. This should therefore be abbreviated as qPCR . A combination of RT-PCR and qPCR is called RT-qPCR.

history

Reverse transcriptases were discovered simultaneously in 1970 by Howard M. Temin in Rous sarcoma virus (RSV) and by David Baltimore in RSV and Moloney Murine Leukemia Virus (MoMLV). For their discovery, they both received the 1975 Nobel Prize in Physiology or Medicine , together with Renato Dulbecco . The generation of cDNA from RNA with the help of reverse transcriptases was first described in 1971. The subsequent amplification of the generated cDNA took place for the first time in 1976 using DNA polymerases. The use of thermostable DNA polymerases took place for the first time in 1989. In 1990, an RT-PCR was carried out for the first time in one reaction batch ( one-step RT-PCR ). The specificity of the reaction could be increased by using hot-start DNA polymerases .

principle

RT-PCR is usually a three-step process: after RNA purification , the RNA is transcribed into DNA, then parts of the DNA are specifically amplified. In order to detect the transcription of a gene, the transcriptome , a ribozyme, ribonucleoproteins or the genome of RNA viruses, the RNA must be examined. Therefore, a reverse transcriptase (RT) is used first, an RNA-dependent DNA polymerase, with the help of which RNA can be transcribed into cDNA . In a subsequent amplification of DNA by the polymerase chain reaction (PCR), specific thermostable DNA polymerases are used which are DNA-dependent, i. that is, they are unable to amplify RNA. The cDNA can then be used as starting material in a PCR in order to amplify specific sequences from it. Most of the time, a 10-minute heating at 95 ° C is used between reverse transcription and PCR, during which the reverse transcriptase is denatured . The RT-PCR products can be analyzed by gel electrophoresis and then cloned or sequenced .

The reverse transcriptases used today are modified enzyme variants from different retroviruses , such as those of the Moloney Murine Leukemia Virus (MoMLV) or the Avian Myeloblastosis Virus (AMV). The different variants of the enzyme have been modified depending on the manufacturer in such a way that they can generate a higher specificity or better yields, for example the RNase H activity naturally occurring in the enzyme is deleted. Conventional reverse transcriptases of retroviral origin used for RT-PCR , such as AMV and MoMuLV reverse transcriptase, are not thermostable at 95 ° C. At the lower temperatures of reverse transcription with these enzymes, however, unspecific bindings of primers to the DNA template and secondary structures in the DNA template occur, which can lead to undesired products and prevent the synthesis of the correct product. However, AMV reverse transcriptase can be used up to 70 ° C. For the reverse transcriptase of MoMuLV, more thermostable RNaseH-negative mutants have been described (mutations E69K, E302R, W313F, L435G, N454K). Furthermore, the template specificity of thermostable DNA polymerases was reduced by exchanging the cofactor (divalent magnesium ions) for divalent manganese salts , so that with a DNA-dependent thermostable polymerase, RNA could also be used in an RT-PCR as a template for the synthesis of DNA. Since the synthesis rate of Taq polymerase with manganese ions was relatively low, the Tth polymerase was increasingly used in this variant of RT-PCR . However, the addition of manganese ions also increased the number of defective products and increased the amount of template DNA required, which is why these enzymes are rarely used for reverse transcription today. These problems could be avoided with the thermostable 3173 polymerase made from thermophilic bacteriophages , which withstands the high temperatures of a PCR for a long time and prefers RNA as a template.

As an RNA-dependent DNA polymerase, the reverse transcriptase requires a short piece of DNA, a so-called primer , to initiate the synthesis of complementary DNA (cDNA). To analyze mRNA carrying poly-A , a so-called oligo-d (T) primer is used here, i.e. several thymine bases which are complementary to the poly (A) tail at the 3 'end of the mRNA .

Very short RNA molecules such as mature microRNAs are much too small (17–22 bases) for the use of conventional primers. Therefore, for the reverse transcription of these nucleic acids, special loop primers are used which hybridize with less than 10 bases only at the 3rd end and thus selectively circumscribe mature microRNAs (instead of mRNAs).

Gene- specific primers are only used in the second step of the RT-PCR . In a modified variant, the One-Step RT-PCR , gene-specific primers are used instead and both reactions are carried out one after the other in the same vessel. With Zero-Step RT-PCR, there is also no isothermal intermediate step that is otherwise carried out during reverse transcription and before the PCR reaction. Due to the high thermal stability of the biotechnologically modified enzyme, both reactions can take place in parallel in the same vessel. At the same time, secondary structures of the RNA are permanently broken up due to the higher temperature of over 55 ° C. Another variant of RT-PCR is RACE-PCR .

Applications

Since a cDNA is complementary to the original mRNA, the amino acid sequence of a protein for which this mRNA codes can also be derived from the genetic code . Since an mRNA in eukaryotes has already been modified and spliced after its transcription , it is also intron- free in contrast to the gene . In addition, this cDNA also enables information to be obtained about whether the associated gene is expressed in different isoforms , i.e. i.e., the mRNA is alternatively spliced . RT-PCR can be used to specifically detect gene expression. RT-PCR is also used to diagnose RNA viruses in blood serum, such as HIV and, more recently, often in connection with influenza A / H5N1 and SARS-CoV-2 .

In the case of a Northern blot , the hybridization probes can be produced by RT-PCR. To analyze the transcriptome , the entire RNA is transcribed into cDNA and copied in an RT-PCR with a mixture of short primers (English random hexamers ). This is usually followed by a microarray or sequencing of the cDNAs. Here, the determination of Expressed Sequence Tags (EST) is often sufficient to identify the transcripts.

literature

Individual evidence

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  2. Baltimore D: RNA-dependent DNA polymerase in virions of RNA tumor viruses . In: Nature . 226, No. 5252, June 1970, pp. 1209-11. doi : 10.1038 / 2261209a0 . PMID 4316300 .
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  4. A. Efstratiadis, FC Kafatos, AM Maxam, T. Maniatis : Enzymatic in vitro synthesis of globin genes. In: Cell (1976), Vol. 7 (2), pp. 279-88. PMID 60178 .
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  7. LN Sellner, RJ Coelen, JS Mackenzie: Reverse transcriptase inhibits Taq polymerase activity. In: Nucleic Acids Res. (1992), Vol. 20 (7), pp. 1487-90. PMID 1374554 ; PMC 312227 (free full text).
  8. MJ Roth, N. Tanese, SP Goff: Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. In: Journal of Biological Chemistry . Volume 260, Number 16, August 1985, pp. 9326-9335, PMID 2410413 .
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  10. A. Telesnitsky, SP Goff: RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template. In: Proceedings of the National Academy of Sciences . Volume 90, Number 4, February 1993, pp. 1276-1280, PMID 7679498 , PMC 45855 (free full text).
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