Primer extension
![Overview of the primer extension. Transcript of interest has uracil at +1 (unknown before assay). 2) Synthesize an 5’ end-labeled primer (using [γ32P] ATP. 3) Make cDNA by extending primer with reverse transcriptase to 5’-end of transcript. 4) Result is a radioactively labeled extended primer cDNA that is denatured and separated on a polyacrylamide gel and detected by autoradiography. Typically, a sequencing ladder using the same primer is also on the gel, allowing for rapid identification of the +1 nucleotide (shown in purple.)](/Images/godic/202502/04/Primer_Extension_Assay0511.jpg")
Primer extension is a technique whereby the 5' ends of RNA can be mapped.
Primer extension can be used to determine the start site of transcription (the end site cannot be determined by this method) by which its sequence is known. This technique requires a radiolabelled primer (usually 20 - 50 nucleotides in length) which is complementary to a region near the 3' end of the mRNA. The primer is allowed to anneal to the RNA and reverse transcriptase is used to synthesize cDNA from the RNA until it reaches the 5' end of the RNA. By denaturing the hybrid and using the extended primer cDNA as a marker on an electrophoretic gel, it is possible to determine the transcriptional start site. It is usually done so by comparing its location on the gel with the DNA sequence (e.g. Sanger sequencing), preferably by using the same primer on the DNA template strand. The exact nucleotide by which the transcription starts at can be pinpointed by matching the labelled extended primer with the marker nucleotide, who are both sharing the same migration distance on the gel.