Primer Extension

Learn about primer extension.

Primer extension increases the accuracy in the ability to map the end of a transcript from the 5’ end to the very nucleotide.

How Primer Extension Works

First we have transcription which usually occurs naturally in vivo. We simply harvest cellular RNA containing the transcript whose 5’ end we want to map.
Then we hybridize a labeled oligonucleotide (the primer) of at least 12 nucleotides to the cellular RNA. Usually primers of at least 18 nucleotides long are used. In order to design a primer we need to know the sequence of at least part of the RNA we want to map. The specificity of this method derives from the complementarity between the primer and the transcript, just as the specificity of S1 mapping comes from the complementarity between the probe and the transcript. In principle this primer should be able to pick out the transcript we want to map from a sea of other unrelated RNAs.
Then we use reverse transcriptase to extend the oligonucleotide primer to the 5’ end of the transcript. The reverse transcriptase makes a DNA copy of an RNA template. Once this primer extension reaction is complete, we can denature the RNA-DNA hybrid and electrophorese the labeled DNA along with markers on a high-resolution gel such as the ones used in DNA sequencing. It is convenient to use the same primer used during primer extension to do a set of sequencing reactions with deoxynucleotides. Then we can use the products of these sequencing reactions as markers.

Primer extension can also give us an estimate of the concentration of a given transcript. The higher the concentration of our transcript, the more molecules of labeled primer will hybridize and therefore the more labeled reverse transcripts, the darker the band on the autoradiograph of the electrophoretic gel.

Factors for a Successful PCR

Can't Find Your Problem? Make sure you see the PCR Forum for PCR troubleshooting and solutions to common problems. You can also post questions to other researchers by simply registering and then clicking on Post New Thread or the question mark below!

PCR Articles

Factors for Successful PCR

PCR Based Site Directed Mutagenesis

Analysis of PCR Reactions

Real Time PCR

PCR Forum Topics

direct sequencing what is the importance of the intensity of the peaks after sequencing of pcr peroducts why are some with sharp peaks while others are not so and why...
stability of 5' and 3' ends to prevent false priming Hi, I am new to PCR and am learning it from the documents I found on the web. In many places I encounter that the 5' end of the PCR primer should...
PCR Smearing i'm currently working on a project which involves nested pcr. the 2 sets of primers worked fine initially, giving distinct bands. however, when we...
basic question Hi, this is very basic question....i am isolating dna from crude fermentation broth and i am trying to prove that organisms coming under lactic acid...
No PCR product for gene os interest HI, i've been trying to get expression of certain genes after stimulating cells with TNF-alpha, but in my PCR i'm only getting the beta actin...
You must REGISTER NOW to post a question in the PCR Forum by clicking here.