LA-PCR

Martin Kennedy mkennedy at chmeds.ac.nz
Thu Dec 9 22:01:05 EST 1993


In article <2e2c2k$63a at mserv1.dl.ac.uk>, unknown at dl.ac.uk writes:
> I have a question regarding RT-PCR. Currently (for the last
> couple of months, that is) I am trying to clone a number of cDNAs
> using the `ligation-anchored PCR' method, as described by Troutt
> et al., PNAS 89, 9823-9825 (1992). I do get lots of PCR product,
> but they are only very small (around 250-300 bp) instead of say
> 800 as expected from sequence data. The method is designed to use
> T4 RNA ligase to add an anchor primer to the 3' end of the 1st
> strand cDNA (i.e. the 5' end of the mRNA), and perform PCR using
> the complimentary sequence as one of the primers. Could anybody
> give some hints as to what might cause the truncated product to
> be formed? 
> Han Schilthuis, Utrecht, the Netherlands 

If you have a GC-rich region, or a region that otherwise forms stable 
secondary structure, the RT may simply not be copying full-length cDNA.  I've
had the same problem with two different genes, using anchored PCR.  You may
need to use a thermophilic RT, or play around with the RT conditions a little.
Why don't you clone and sequence what you are getting, and make another primer
perhaps closer in to the problem region, that may give you a better chance of
getting the more 5' regions?

Good luck,
          
Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
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