Sequencing with Degenerate primers?

Ron Kagan rkagan at ewald.mbi.ucla.edu
Sun May 15 16:38:23 EST 1994


In article <01HC93XRX67M001U1X at GW.AGR.CA> , SCOTTA at NCCCOT7.AGR.CA writes:
>Hi there,
>
>I've been using a thermocycle sequencing kit for sequencing a PCR product
>derived from a couple of potyvirus strains and was wondering if anyone
had
>any success using degenerate primers for sequencing? The primer I intend
to
>use has only a 4-fold degeneracy, and was wondering if it was suitable
to use
>for sequencing. If anyone could give me some advice, it would be really 
>appreciated.
>
>Thanks in advance,
>
>Andrew Scott
>======================================================================
======
>Agriculture Canada - Central Plant Health Laboratory
>3851 Fallowfield Road
>Nepean, Ontario
>CANADA   K2H 8P9			Email: scotta at ncccot7.agr.ca
>======================================================================
======


I've sequenced PCR products with the degenerate primers (up to 512X
degenerate) used to amplify the products.  The higher the degeneracy of
the sequencing primer, the lower the effective concentration of the
primer that can bind to the template, so I was using about 2.5-5 pmole
(instead of 0.5 pmol for non-degenerate primers) and 32-P end labelling
of primers in cycle sequencing reactions.  

However, for something that is only 4X degenerate, normal
cycle-sequencing conditions  with end-labelled primers would probably
work fine!

Ron Kagan
rkagan at ewald.mbi.ucla.edu

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