Sequencing high G + C DNA

Sherri Fraser sfraser at acs5.acs.ucalgary.ca
Tue May 2 16:29:23 EST 1995


Hi,
	It sounds like you have a couple of problems.  The first
is getting your reactions to sequence through the GC rich region.
I know Sequenase used to have a sequencing kit for high
temperatures, using Taq DNA polymerase.  I believe they called it
"TAQuence".  I don't know if they still have this kit.  It uses
Taq, so the reactions are run at higher temps, thus allowing
sequence through regions of high secondary structure.
	The other problem is one I have dealt with, and that is
how to see these GC-rich sequences on a gel, even if the enzyme
could sequence through them.  CURRENT PROTOCOLS (the red book)
has a great procedure for formamide-containing sequencing gels
that I have used to sequence through a very stable secondary
structure.  You may want to try this.  This will only work if the
enzyme could make it through.  It seems to do a good job of
eliminating compressions.

Sherri Fraser
The University of Calgary


Allen Gathman (C741SCB at SEMOVM.SEMO.EDU) wrote:
: In article <3o5dk4$rv3 at synapse.bms.com> Steven Goldberg <goldberg at bms.com> writes:
: >I have been attempting to sequence DNA of high G + C content (ca. 72%)
: >.

: (snip, snip)

: We have similar problems.  Although we haven't yet figured it out, we
: are about to try the suggestions in a paper titled "General method for
: amplifying regions of very high G+C content" from NAR 21:2953-2954.

: Basically, the authors say to use denaturation temp of 98 C, five min
: combined annealing/elongation at 70 C, and to try reducing template
: concetntration.  (Obviously, I'm assuming you're using a cycling
: sequencing protocol here.)

: We're about to try this; will let you know.  I'd appreciate hearing any
: other suggestions.

: ************************************************************************
: |    Allen Gathman                |                                    |
: |    Biology Department           |  Internet: C741SCB at SEMOVM.SEMO.EDU |
: |    Southeast Missouri St. U.    |  Phone: (314) 651-2361             |
: |    Cape Girardeau, MO 63701     |  Fax: (314) 651-2223               |
: ************************************************************************
: |    "Anyone would know that," said Dorothy.                           |
: |    "Certainly; that is why I know it," returned the Scarecrow.  "If  |
: |    it required brains to figure it out, I never should have said it."|
: |    -L. Frank Baum                                                    |
: ************************************************************************




More information about the Methods mailing list