5' end of cDNA

Frank A. Cantone fac2 at cornell.edu
Thu Feb 10 14:03:22 EST 1994


Dear Netters

I have used degenerate primers and PCR to obtain  a "core" cDNA, and RACE
PCR to obtain the 3' end of my cDNA.  I have, for the past several months,
tried to use  RACE to get the 5' end, but without any success.  In fact, it
has been a nightmare.  The few times that I have been fortunate to get
amplification products, when cloned and sequenced, exhibit only 50% to 60%
similarity to the overlap region of the core cDNA.  I have wasted a lot of
time and I am ready to try another approach.

I don't have a library from which to screen and my RNA is in low abundance
(the primary considerations for using PCR in the first place).  One
approach that has been suggested is to design a primer (# of bases?) to the
5' end, anneal it to my RNA, use reverse transcriptase to extend, and clone
(essentially cDNA synthesis).  Since my RNA is in low abundance it has also
been suggested that I try and perform a solution hybridization between my
RNA preparation and single stranded plasmid clone of the "core" cDNA to
enrich for my RNA.  Then "melt off" the RNA and use that for the cDNA.  Is
this a viable approach?  Are there any other alternatives (besides RNA
sequencing)?  Any advice at all would be greatly appreciated.  Thank you.

Frank A. Cantone
fac2 at cornell.edu  



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