Primer extension is primarily used in molecular biology to determine the start site of RNA transcription for a known gene.
Understanding Primer Extension
This molecular technique provides a precise method for researchers to map where the transcription process begins on a DNA template. Knowing the exact transcription start site (TSS) is crucial for understanding gene regulation, as it often corresponds to promoter regions and influences how and when a gene is expressed.
How the Technique Works (Based on Reference)
The process relies on extending a short DNA sequence (a primer) along an RNA template. According to the provided reference, this technique:
- Requires a radiolabelled primer: The primer, typically 20 to 50 nucleotides long, is synthesized and tagged with a radioactive isotope. This label allows the extension product to be detected later.
- Uses a primer complementary to the gene's 3′ end: The primer is designed to bind specifically to a region near the 3′ end of the messenger RNA (mRNA) transcribed from the target gene.
- Involves reverse transcription: Once the primer binds to the RNA, an enzyme called reverse transcriptase is used. This enzyme synthesizes a complementary DNA (cDNA) strand, starting from the 5' end of the primer and extending towards the 5' end of the RNA molecule.
- Determines the start site: The length of the resulting cDNA product corresponds to the distance between the primer binding site and the 5' end of the RNA. By measuring the length of this radiolabelled cDNA product (usually via gel electrophoresis) and knowing the location where the primer annealed, the 5' end of the RNA (and thus the transcription start site) can be determined relative to the known gene sequence.
In essence, primer extension acts like a molecular ruler, measuring the distance from a known point within the RNA (where the primer binds) back to its beginning (the transcription start site).
