INVERSE-PCR

Paul N Hengen pnh at ncifcrf.gov
Fri Jan 10 16:03:31 EST 1997


Dr Alok Adholaya (aloka at teri.ernet.in) wrote:

: Dear Netters,
: 
: I am trying to clone a gene from fungus of intrest  using a
: degenerate primer strategy. Now the total size of the gene is near
: about 1kb, and my primers have been designed to amplify around
: 280bp of the  C terminal region of the gene.  I am able to get the
: expected size product thus the  degenerate primers work. The
: sequencing is on now. 
: 
: The challange is to get the whole gene, which could be done by
: making a probe of the 280bp fragment and doing a southern, but alas
: this cannot be done as the fungus which i work on is available in
: miniscule quantity thus getting enough DNA for southern or making
: a genomic library to screen out is ruled out.
: 
: Could anyone suggest any PCR based strategy so that i could fish
: out the whole gene. Now Inverse-PCR is one which comes to my
: mind.....................

Have you considered boomeranging it in both directions? You might pick
a good long primer from the known sequence and go from there. You could
get the primer biotinylated and then extract the sequence to be amplified
away from the bulk using paramagnetic particles. You could then manipulate
the ends to create blunts and ligate boomer-ends onto it -> then Hoola! :-)

--
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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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