Sequencing with hexamers (was degenerate primers)

Richard T. Pon rtpon at acs.ucalgary.ca
Mon Oct 17 14:48:19 EST 1994


In article <cupton.1132543104B at ra.uvic.ca> cupton at sol.uvic.ca ( Chris Upton) writes:
>Hi,
>  Does anyone have a reference to sequencing with Sequenase (ds DNA) using
>degenerate primers? I was going to try the method used for multiple short
>primers. Can't find the reference.....
>
>Is it simply, anneal and extend+label on ice, then finish with ddNTPs at 37C?
>
>It would save a bunch of $$.
>Cheers,
>   Chris

Are you refering to the method of primer walking which uses
contiguous strings of short (i.e. hexanucleotide) primers? If so
the following references will help:

1, J. Kieleczawa, J.J. Dunn, and F. W. Studier, 1992, DNA sequencing by
primer walking with strings of contiguous hexamers, Science V258,
p1787.

2, N.S. Ghiso, H. Parekh, and G.G. Lennon, 1993, A subset of 1200
hexamers is sufficient to sequence over 95% of cDNAs by hexamer
string primer walking, Genomics V17, p798.

3, W. R. McCombie and J. Kieleczawa, 1994, Automated DNA
sequencing using 4-color fluorescent detection of reactions
primed with hexamer strings, Biotechniques V17, p574.

Our laboratory has already synthesized the 1200 hexanucleotides
described by Ghiso et. al. We have a limited number of these sets
which will be available as an experimental set as soon as I finish
writing a small computer program which will make finding priming
sites easier. Each set contains 2.5 nmol of each primer in
individually packaged tubes.
	Anyone interested in these primers should contact me at
either rtpon at acs.ucalgary.ca, tel: 403-220-4277, or fax:
403-283-4907.

Richard T. Pon
University Core DNA Services
University of Calgary
Calgary, AB, Canada



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