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  Answer ID  
1
  Product  
Tech Service: Amplification & Primers
  RT-PCR, cDNA, and RACE Systems
  Date Created  
06/10/1999 04:31 PM
  Date Updated  
09/12/2002 03:50 PM
  Access Level  
Everyone

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  How should I prime my cDNA?
  Question
  For first strand synthesis of cDNA, is it better to prime with oligo(dT), random hexamers, gene specific primer (GSP) or combination of these primers?
  Answer
  It depends on your experimental goals. Oligo(dT) is used for many systems and is key for full-length cDNA. Random hexamers give a series of short first-strand products spanning the entire mRNA. Use of random hexamers may be helpful if your PCR fragment is at the 5 end of a large mRNA. To ensure representation of the 3 end, oligo(dT) can be added along with random hexamers. Gene specific primers (GSP) for cDNA synthesis may also be used and are required in a few applications such as 5 RACE. In instances of GC-rich templates, or templates rich in secondary structure, a GSP may not work as well as priming with oligo dT for first strand synthesis. If an RT-PCR is problematic, trying the different options of oligo dT, random primers and/or GSP for priming first strand synthesis may find a solution.

References:
FOCUS 12 p 47 available from Invitrogen Technical Services
FOCUS 14 p 91 available from Invitrogen Technical Services
Frohman,M.A., Dush,M.K., Martin, G.R. (1988) <i>Proc. Nat. Acad. Sci</i> USA 85, 8998
 
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