How can we directly sequence PCR-amplified DNA from agarose.

Brian Foley brianf at med.uvm.edu
Tue May 3 12:45:08 EST 1994


: Wasun Chantratita (asmsi002 at CMU.CHIANGMAI.AC.TH) wrote:
: : Dear Netters
: : 	I am now trying to directly sequence PCR-amplified DNAs by cycle 
: : sequencing. I was told that excess nucleotide and amplified primers can 
: : interfere with the sequencing reactions. Thus,  PCR-amplified DNAs must 
: : be purified by either a glass matrix or isopropanol precipitation. 
: : However, would it be possible that we can cut a DNA band from perhaps low 
: : MP agarose and subject to cycle sequencing directly. If yes, would you 
: : please provide me that protocol or reference(s).

: : 	Many thanks in advance
: :         Wasun

	BioTechniques Volume 16, Number 4, April 1994 has several 
articles on direct sequencing of PCR products: On pages 572-573 Barnard,
Puder, Begum and Chen describe conventional Sequenase T7 sequencing of
PCR products using as little as 80 nanograms of PCR product and using THE
SAME PRIMERS used in the PCR.
	They found that 32-P or 33-P was better than 35-S due to the shorter
half-life and higher energy giving a good exposure in less time.
	They gel-purified the PCR product using QIAEX from QIAGEN Corp.

	Other articles spell out how cycle-sequencing is good for 
applications where much less than 80 ng of template DNA is available.
Cycle sequencing is good, but not quite as good as Sequenase, because the
thermostable polymerases lead to uneven band intensities.

--
********************************************************************
*  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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