How the Primer Extension Assay Works
Early cycle decisions are coming out! Good luck to everyone getting decisions soon! (I got one this morning help-)
The primer extension assay helps us determine the shape of an RNA molecule by telling us which regions of the molecule are single-stranded. This helps because RNA often serves various functions in the hairpin/stem loop structure, which involves specific locations of ds and ssRNA, pictured below:
Identifying the intricacies of the RNA’s structure can reveal a lot about its function, just like how finding protein structure is important. Also, this can help find the transcriptional start site as it marks key regions.
The process is as follows:
1) We add a chemical(s) that binds to RNA nucleotides only when they are unbound (thereby marking the ssRNA regions). We strategically calculate concentration of chemical to add, such that only one chemical binds to each RNA strand. Typically, these chemicals don't bind all 4 nucleotides, so we need to create a cocktail such that all 4 nucleotides are targeted and we can accurately conduct this assay.
2) We add a primer to the start of the RNA molecule and create a complementary DNA strand using reverse transcriptase, which does the opposite of regular transcription: it creates complementary DNA (cDNA) from RNA instead of the normal RNA from DNA.
3) This cDNA strand will stop being created wherever the chemical is, as the chemical bound to the base is a modification that prevents reverse transcription.
4) Collect these cDNAs of various lengths and do gel electrophoresis with them to see their sizes.
5) Now we need to interpret the gel. To do this, we need to understand the whole RNA. Take intact RNA molecules and reverse-transcribe them to get full cDNAs.
6) Conduct Sanger sequencing on these cDNAs to get another gel. This is your "DNA marker" gel. (If you don't know how Sanger works / how to interpret a Sanger gel, read this.)
7) Now you have two gels: the full RNA marker one with controlled steps, and the experimental RNA one to help us find where the RNA is ss or ds.
Let's talk about how to interpret the gels. The full RNA marker/Sanger one is read from bottom to top, and this will give you not the cDNA sequence, but rather the sequence of the strand complementary to the cDNA (so the sequence on the gel is the RNA sequence, but with thymines instead of uracils). The actual primer extension gel, however, reflects the cDNA's sequence, so it's complementary to the actual RNA sequence. This gel also needs to be read from the bottom to the top (smaller fragments go to the bottom of the gel through all the holes and stuff, and smaller fragments are earlier in the sequence). To get the RNA sequence of this gel, you need to find the complementary DNA sequence to what is displayed on the gel, and replace the thymines with uracils. Another quirk: you need to shift every band on the gel 1 unit up relative to the Sanger gel because the primer extension stops 1 nucleotide before the marked one, so the length is 1 unit shorter than recorded on the gel. Then you compare bands between the gels to see where the RNA is single-stranded (marked by bands).
Here's a cool video about this:
And this goes more into detail about how this method can be used for transcription start site mapping purposes: mine was focused on ss vs ds differentiation for hairpin mapping: https://en.wikipedia.org/wiki/Primer_extension.
