Primer extension

Primer extension is a technique whereby the 5' ends of RNA can be mapped - that is, they can be sequenced and properly identified.

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.

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.

The hybridization probe for primer extension is a synthesized oligonucleotide, whereas S1 mapping requires isolation of a DNA fragment.

Unlike S1 mapping, however, primer extension can only be used to locate the 5’-end of an mRNA transcript because the DNA synthesis required for the assay relies on reverse transcriptase (only polymerizes in the 5’ → 3’ direction).

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.)