Degenerate PCR - Sequencing?

Brian Foley brianf at med.uvm.edu
Wed Mar 9 15:37:43 EST 1994


Jim Gerlach (GERLACHJ at QUCDN.QueensU.CA) wrote:
: We have been doing PCR with degenerate primers (17mers, usually 256 fold) 
: and obtaining products in the 150-300 bp size. After doing restriction 
: digests to establish that we had produced some non-human products we are
: now 
: ready to sequence. We could, of course, clone and sequence in a 
: conventional 
: fashion but I'm wondering what are the chances of doing sequencing using 
: the 
: degenerate primers. Anyone tried this (or some other nice trick)? Thanx for 
: any sugguestions or help.
:  
	I have directly sequenced a few PCR products, sometimes using the 
same primer I used to amplify the fragment, and sometimes an internal 
primer, sometimes end-labelling the primer and sometimes incorporating 
35-S during the sequencing.
	The best results were with an internal primer (not used in the 
PCR) that was end-labelled.  But several other factors are important too, 
such a how pure is the PCR product (gel isolated is better than just 
using ultra-filtration to remove primers).  Cycle-sequencing is often 
needed because the amount of template is often quite low.
	Overall, directly sequencing a PCR product is not an easy thing 
to do well, but it is such a powerful technique that it can be worth the 
time it takes to get it working.  If you just want to sequence one 
product, clone it.  But if you will be wanting to sequence many such 
products in the future, it is worth it to not have to clone each one.
	Best of luck to you.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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