cloning full-length cDNAs without libraries

Jon Nakamoto jnakamot at pediatrics.medsch.ucla.edu
Mon Jan 16 16:03:00 EST 1995


In the eternal search for the ideal method for cloning full-length
cDNAs, I ran across Clontech's Jan '95 Clontechniques describing
their Marathon cDNA amplification kit. It is basically a clever
modification of 5' & 3' RACE strategies, but I personally find their
"introductory" price of $425 quite outrageous. They don't cite any
literature references, and I'm curious if anyone knows who
originally developed it (or maybe it's a Clontech original?)

It's hard to describe without diagrams, but in a nutshell: perform
standard 1st & second-strand cDNA synthesis using an NNT(30)
lock-dock oligo-DT primer, then blunt-ligate (onto both ends of your
cDNA) a partially dble-stranded adaptor. This adaptor is unique in
that the non-blunt end has a long 5' overhang (top strand) and a
amine-blocked 3' end (bottom strand). You now have an unamplified
library of adaptor-ligated ds cDNAs. Take an upstream "adaptor"
primer with sequence identical to the 5' overhang region of the
adaptor and a downstream primer with some internal cDNA sequence,
and perform PCR. During the first cycle you get extension of the
downstream primer; then this extension product can serve as template
for exponential amplification. Other cDNAs to which the adaptor was
ligated can't extend because there is no binding site for the
upstream primer in the adaptor, thus decreasing background relative
to classic 5'RACE. If you also do a separate PCR reaction with
another upstream primer which is 5' of the lower primer in the
previous rxn, as well as the adaptor primer (now serving as the
lower primer), you'll get a fragment which contains the 3' end of
your cDNA and overlaps the 5' fragment from the previous rxn. You
can anneal and extend these fragments to get a full-length cDNA,
which can then be amplified from the mixture of DNA strands by a
long-distance PCR rxn using the adaptor primer as the upstream
primer and the original NNT(30) cDNA primer (for some reason the
adaptor primer alone won't amplify the whole cDNA as well). TA clone
it or design the adaptor primer & NNT(30) primer to include
restriction sites for cloning. 

It sound promising to me, but I can't see paying through the nose
for a kit which doesn't even include the long-distance PCR enzymes.
Your comments (personal experience, references, questions) are
welcome.

thanks,
Jon

Jon Nakamoto
Clinical Instructor of Pediatrics/Endocrinology, UCLA
jnakamot at pediatrics.medsch.ucla.edu



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