Reverse transcription polymerase chain reaction
The use of the reverse transcriptase (RT) and polymerase chain reaction (PCR) - - reverse transcriptase - polymerase chain reaction (RT- PCR), the combination of two methods of molecular biology is to demonstrate RNA, such as the expression of specific genes in cells, tissues, and blood serum or ribozymes, ribonucleoproteins, or the genome of RNA viruses. Used RT- PCR in research and diagnostics.
The abbreviation RT- PCR sometimes known the real time quantitative PCR, which can lead to confusion. This should therefore be abbreviated with qPCR. A combination of RT-PCR and qPCR is called qRT- PCR.
History
Reverse transcriptases were discovered in 1970 simultaneously by Howard Temin in Rous sarcoma virus (RSV) and David Baltimore, RSV and the Moloney murine leukemia virus ( MoMLV ). For their discovery both received the 1975 Nobel Prize in Physiology or Medicine, together with Renato Dulbecco. The generation of cDNA from the RNA using reverse transcriptase was first described in 1971. The subsequent amplification of the cDNA was generated in 1976 using DNA polymerases. The use of thermostable DNA polymerases was performed for the first time in 1989. In 1990, for the first time, an RT -PCR in a reaction mixture (English One-Step RT -PCR) was performed. The specificity of the reaction could be increased by the hot-start DNA polymerases.
Conventional RT-PCR for reverse transcriptases used retroviral origin, such as the AMV and MoMuLV reverse transcriptase, are not thermostable. At the lower temperatures, a reverse transcription with these enzymes, however, non-specific binding of primers to the DNA template and secondary structures in the DNA template occur, which lead to undesired products and can prevent the synthesis of the correct product. Therefore, the template specificity of thermostable DNA polymerase has been reduced to divalent manganese salts by exchange of the cofactor ( divalent magnesium ions ), so that with a DNA dependent thermostable polymerase and RNA could be used in an RT-PCR as a template for the synthesis of DNA. Because the rate of synthesis of Taq polymerase was relatively low with manganese ions, the Tth polymerase was used increasingly in this variant of RT-PCR. However, the addition of manganese ions also increased the number of defective products and increased the necessary amount of template DNA, which is why these enzymes are now rarely used for reverse transcription. These problems could be avoided by using the thermostable polymerase 3173 from thermophilic bacteriophage which withstands the high temperature of a PCR for a long time and preferably as a template RNA.
Principle
RT-PCR is a generally two-step process: First, the RNA is transcribed into DNA, then increased specific parts of the DNA. To detect the transcription of a gene of the transcriptome, a ribozyme, or ribonucleoproteins of the genome of RNA viruses, the RNA to be studied. Therefore, a reverse transcriptase (RT ) is first used, an RNA-dependent DNA polymerase, with 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.e. they are not able to amplify the RNA. The cDNA can be used as starting material in a PCR, following to amplify specific sequences from this. This denatures the reverse transcriptase.
The products of RT- PCR can be analyzed by gel electrophoresis and subsequent cloning or sequencing.
The reverse transcriptases are used today are altered enzyme variants from different retroviruses such as the Moloney murine leukemia virus ( MoMLV ) or Avian myeloblastosis virus ( AMV ). The different variants of the enzyme have been modified according to the manufacturer so that they can generate a higher specificity or better yields, for example the naturally occurring enzyme in the RNase H activity is deleted.
As an RNA -dependent DNA polymerase reverse transcriptase requires a short piece of DNA, called a primer, to initiate the synthesis of the complementary DNA (cDNA). For the analysis of poly-A mRNA Carrying a so-called oligo -d is used here (T ) primer, that is more thymine bases, which comprises (A ) tail are complementary to the poly on the 3 'end of the mRNA.
Very short RNA molecules such as microRNAs mature are much too small ( 17-22 bases ) for the use of conventional primers. Therefore, special sanding primers are used for reverse transcription of nucleic acids with less than 10 bases hybridize to the 3 'end and so only selectively mature microRNAs (instead mRNAs ) rewrite.
Only the second step of the RT-PCR Gene specific primers are used. In a modified version, which will be one-step RT- PCR instead directly used gene -specific primers and both reactions are carried out in succession in the same vessel. A further variant of RT-PCR, the RACE -PCR.
Applications
Since a cDNA complementary to the original mRNA, including the amino acid sequence of a protein can be derived from this on the basis of the genetic code, for encoding this mRNA. Since an mRNA has been modified and spliced in eukaryotes after their transcription, it is also intron - free in contrast to the gene. In addition, this cDNA allows also to obtain information about whether the corresponding gene is expressed in different isoforms, that is, the mRNA is alternatively spliced. About RT- PCR thus allows you to specifically detect gene expression. RT-PCR is also used in the diagnosis of viral RNA in the blood serum, such as HIV, and more recently also often associated with influenza A/H5N1.
In a Northern blot hybridization probes by RT- PCR are produced. For the analysis of the transcriptome, the total RNA is used in an RT- PCR with a mixture of short primers ( engl. random hexamers ) can be rewritten and copied into cDNA. Following usually takes place a micro array or a genome sequencing (in this case only of the cDNA ).