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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

  1. 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. 
  2. 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.
  3. 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.

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