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IPCR

Dick Beeman beeman at crunch.usgmrl.ksu.edu
Fri Jul 29 16:00:33 EST 1994


Lorant Lakatos asked about Inverse PCR:

> Subject: IPCR
> From: Lorant Lakatos, lakatos at hubi.abc.hu
> >Dear Netters, 
> >I am looking for a good detailed protocol for
> >inverse-PCR. I have tried  some but I did not get 
> >any bands.
> >Thanks so much
> >   Lorant

To get junction fragments beyond a region of known sequence, universal PCR
can be used as an alternative to inverse PCR.  We have used this approach
to clone  insertion junctions of a retrotransposon.  The method is based on
one described by Sarkar et al. in PCR Methods & Applications 2:318-322
(1993), and worked beautifully for us on the first try.  Briefly, you need
to make two nested "forward" primers based on a region near the end of your
known sequence, with the 3' ends pointing out toward the unknown flanking
region.  The universal "reverse" primer has the following design:
5'-T7-X-NNNNNNNNNNGATC-3', where T7 is the phage promoter sequence, or any
other specific sequence not present in the template DNA, X is an optional
restriction endonuclease recognition sequence to facilitate subsequent
cloning of the amplified fragments (we omit this, since we do TA cloning),
the next 10 nt's are completely degenerate, and GATC is any specific 4-6 nt
sequence (the restriction recognition sequence could be put here rather
than next to T7).  Do two rounds of nested PCR, the first using forward
specific primer 1 and the universal reverse primer.  Then take a 1 µl
aliquot as the template for the second round of PCR, this time using
forward primer 2 and T7 as reverse primer.  Gel-purify the individual bands
(you will probably get several bands from each reaction), clone and
sequence, using forward specific primer 2 as sequencing primer.  Good luck.
-- 
Dick Beeman
beeman at crunch.usgmrl.ksu.edu
"One of the advantages of being disorderly is that one is constantly making
exciting discoveries".  A. A. Milne



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