Marc A. Goldstein
magoldst at ix.netcom.com
Wed Dec 11 22:17:24 EST 1996
In article <58mo4t$c0l at infoserv.rug.ac.be>, Els.VanGeldre at rug.ac.be
> I'm wondering wether it is possible to perform cycle sequencing reactions
> directly on genomic DNA with a specific primer. I would like to use this
> technique in order to isolate a gene of about 2 kb (in comparison with
> similar genes). My problem is that I only have very little information
> (about 100bp) on the sequence of this gene.
> Anyone has experience with this ?
> Tnx for answering,
Another option if your direct sequencing does not work (I've not
had good success with this in the past) is to use the technique of
inverse PCR to amplify the region of interest. If you have 100 bp of
sequence, that is enough to design two OUTWARD facing primers. These
primers can be used to amplify the flanking sequences on either side of
that 100 bp, after the genome is restricted with an enzyme such as
HindIII, then circularized via ligase. This is a neat technique that I
have used in cases like this, and it works very well. It will take about
two lab days to go from genomic DNA to purified PCR fragment, ready for
sequencing. Check out any of the PCR books for more details. I like the
PCR Protocols one (dated but still useful).
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