cycle sequencing

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 
says...
> Hi,
> 
> 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,
> 
> Els 
> 
> 
Els,

	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).

	Good luck!

	Marc Goldstein



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