Primer Extension

Primer extension [ pʁaɪ̯mɐ ] [ ɪkstenʃn ] is a molecular biological technique, which is generally used for determining the base sequence of the 5'-end of the mRNA. To the 5 'ends of a specific RNA species are calculated from a mixture of RNA molecules. The 5'-end is usually a transcription starting point of the subject gene.

Description of the method

The so-called primer - - In the primer extension a short, (mostly ) synthetically produced DNA oligonucleotide is annealed to previously isolated RNA. To the RNA and the primer are combined and heated. By heating to double-stranded regions of the RNA separated. Subsequently, the mixture is cooled again so that the primer can anneal to the complementary base sequence of the RNA itself. This is the primer in the RNA mixture, the corresponding gene.

The system comprises two additional components substantially. Are the deoxyribonucleoside triphosphates (dNTPs, short dATP, dCTP, dGTP and dTTP ) which serve as nucleic and an enzyme is referred to as reverse transcriptase. Reverse transcriptase binds to DNA / RNA hybrid molecule and fills the primer at the 3 ' end with nucleotides. The mRNA of the observed gene serves as a template. By lengthening (extension) of the primers to the cDNA. The cDNA can be subjected ( by alkaline lysis and / or RNA - degrading enzymes, for example ) of a further analysis, after separation of the RNA.

In general, the further analysis is in progress so that the cDNA is compared from the primer extension approach, a sequencing approach already known chromosomal DNA. The well-known, most cloned piece of DNA containing the primer region and should extend to the " other side" will not yet precisely mapped "front" end of the gene. The sequencing reactions are performed with the same primers as the cDNA synthesis. Primer extension products and the reactions of the DNA sequencing can be applied to a so-called " sequencing " and subjected to electrophoresis. On the basis of comparison of the DNA fragment length of the transcription start point can be determined.

Formation

Primer extension was developed in the late 1970s. Here, two main directions have been followed and merged: DNA sequencing by partial chain termination and cDNA synthesis by reverse transcriptases. During primer extension was initially used as a phrase rather than as a method name, the term set by the beginning of 1980 as a technical term and refers mostly the variant described above. Methods in which the sequencing are coupled by partial chain-termination directly to the cDNA synthesis, are now referred to, rather than direct RNA sequencing.

Homonyms and synonyms

Homonyms: In addition to some of primer extension different molecular genetic methods, which also plays the extension of an oligonucleotide by nucleic acid polymerases play a role, the phrase " ... primer extension ... " especially in the description of DNA replication is used.

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