5' cDNA question

Martin Kennedy mkennedy at chmeds.ac.nz
Sun Jul 31 05:03:51 EST 1994


In article <1994Jul28.090903.1 at opal.tufts.edu>, slee at opal.tufts.edu writes:
> Dear netters:  does anyone one know a sure fire method of determining the 
> 5' end of a cDNA.  How do you know whether the site you map by primer ex-
> tension, s1 nuclease is the TSS or an intron?  This is especially hard
^^^^^^^^^
> to figure out in TATA-less receptor cDNA's  I would appreciate any
> ideans and opinions.  Steph e-mail "slee at opal.tufts.edu"  Thanks.

Primer extension isn't going to be affected by the presence of introns - you 
are extending a cDNA molecule on an RNA transcript that has already been 
spliced.  Where primer extension, and other RT based methods (like RACE), 
falls down is if the template has unusual or strong secondary structure - 
particularly large CG-rich tracts.  Normally you should use two quite 
independent methods to determine the 5' end, like primer extension plus S1 
nuclease mapping.  If they give the same or similar answers, you're probably 
in the right ballpark. 

Cheers,

Martin

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