Primer extension

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The primer extension [ˊpʁaɪ̯mɐ] [ɪkˊstenʃn] is a molecular biological method which is generally used to determine the base sequence of the 5 'end of the mRNA . For this purpose, the 5 'ends of a certain RNA species are determined from a mixture of RNA molecules. The 5 'end is mostly also a starting point for transcription of the gene under consideration .

Description of the method

When primer extension is a short (usually) synthetically produced DNA - oligonucleotide - the so-called primer - attached to previously isolated RNA. To do this, the RNA and the primer, which was usually radioactively labeled with T4 polynucleotide kinase , are combined and heated. Double-stranded areas of the RNA separate as a result of the heating. The batch is then cooled again, so that the primer can attach to the complementary base sequence of the RNA. In this way, the primer finds the corresponding gene in the RNA mixture.

The system essentially contains two further components. These are the deoxyribonucleoside triphosphates (dNTPs, dATP, dCTP, dGTP and dTTP for short), which act as nucleobases and an enzyme called reverse transcriptase . The reverse transcriptase binds to the DNA / RNA hybrid molecule and fills the primer at the 3 'end with nucleotides. The mRNA of the gene in question serves as a template. The extension turns the primer into a cDNA . After separation from the RNA (e.g. by alkaline lysis and / or RNA-degrading enzymes ), the cDNA can be subjected to further analysis.

In general, the further analysis runs in such a way that the cDNA from the primer extension approach is compared with a sequencing approach of already known genomic DNA. The known, mostly cloned piece of DNA contains the primer area and should extend on the "other" side beyond the not yet precisely mapped "front" end of the gene in question. The sequencing reactions are carried out with the same primer as the cDNA synthesis. The primer extension products and the DNA sequencing reactions are applied to a so-called “sequencing gel” and subjected to electrophoresis . The starting point of transcription can be determined by comparing the lengths of the DNA fragments.

Emergence

The primer extension was developed in the late 1970's. Essentially two directions were pursued and combined: DNA sequencing by partial chain termination and cDNA synthesis by reverse transcriptases. While primer extension was initially used as a group of words rather than the name of a method, the term became established in the early 1980s as terminus technicus and mostly describes the variant described above. Methods in which the sequencing is linked directly to the cDNA synthesis by partial chain termination are now more commonly referred to as direct RNA sequencing .

Homonyms and synonyms

Homonyms : In addition to some molecular genetic methods other than primer extension, in which the lengthening of an oligonucleotide by nucleic acid polymerases also plays a role, the phrase "... primer extension ..." is mainly used to describe DNA replication .

Synonyms : cDNA extension by reverse transcriptase; reverse transcriptase mapping; RT mapping; RT mapping

Individual evidence

  1. ↑ Sanger, F. & Coulson, AR (1975): A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. In: J. Mol. Biol. 94: 441-444. PMID 1100841
  2. ↑ Sanger, F. et al. (1977): DNA sequencing with chain-terminating inhibitors. In: Proc. Natl. Acad. Sci. USA 74: 5463-5467. PMID 271968
  3. ↑ Thompson, JA et al. (1979): Characterization of the 5'-terminal structure of simian virus 40 early mRNA's. In: J. Virol. 31: 437-446. PMID 90173
  4. ↑ Qu, HL et al. (1981): Improved methods for structure probing in large RNAs: a rapid 'heterologous' sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5 'terminal domain of eukaryotic 28S rRNA. In: Nucl. Acids. Res. 11: 5903-5920. PMID 6193488

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