Primer extension

BIOC/MCB 568 -- Fall 2010
John W. Little--University of Arizona

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 Methods

 

Primer extension is used to map the 5' ends of DNA or RNA fragments. It is done by annealing a specific oligonucleotide primer to a position downstream of that 5' end. The primer is labeled, usually at its 5' end, with 32P. This is extended with reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment that ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA templates.

 

Uses:

1. Mapping the 5' end of transcripts. This allows one to determine the startpoint of transcription (assuming the mRNA isn't further processed), which helps localize promoters or TATA boxes.

2. Quantifying the amount of transcript in an in vitro transcription system. An example is the Yudkovsky et al. paper.

3. Determine the locations of breaks or modified bases in a mixed population of RNA or DNA samples. This is useful in applications like footprinting. Two different methods are used. In one, the modified nucleotide cannot be recognized by the polymerase or reverse transcriptase; in such cases, the chain ends at the site of modification (as with KMnO4 -- for instance in the Kainz and Roberts paper). In the other, the modification is converted in a later step of the analysis to a strand break by chemical treatment. For instance, the sites of modifications by dimethyl sulfate (DMS) can be identified by treating DNA with DMS, exposing the sample to conditions that break the backbone at the site of modification, followed by primer extension.

 


BIOC/MCB 568 -- University of Arizona

http://www.biochem.arizona.edu/classes/bioc568/bioc568.htm
Last modified August 18, 2010
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